Introduction

Allogeneic stem cell transplantation (allo-SCT) is the treatment of choice for many hematological malignancies as well as for those with a severe immunodeficiency. However, acute graft-versus-host disease (GVHD), which is the major toxicity of allo-SCT, is a complex process involving various dysregulated inflammatory cytokine cascades and distorted responses of the donor cellular effectors to the host alloantigens.¹ The treatment of acute GVHD is difficult because steroids and other immunosuppressants increase the likelihood of a lethal infection regardless of whether the GVHD has been controlled or not.² Currently, there is no effective therapy for severe steroid-refractory acute GVHD.³ Several drugs and antibodies have been used but with discouraging results. The removal of the donor T cells from the bone marrow graft has been performed, which decreases the incidence of acute GVHD.⁴ Unfortunately, T cell depletion is also associated with an increased incidence of relapse and graft failure.⁵

In addition to hematopoietic stem cells, the bone marrow also contains mesenchymal stem cells (MSCs). Apart from the postnatal marrow, MSCs have also been isolated from adipose tissue and the fetal liver, blood, bone marrow, lung and cord blood.^{6, 7} MSCs have immunomodulatory properties and have been reported to inhibit T cell proliferation in vitro.^{8, 9, 10, 11} Several mechanisms have been proposed to explain how MSCs suppress T cell activation and modulate the immune response. Among them, the suppression seems to be mediated by a soluble factor or factors produced by the MSCs themselves. It was suggested that the soluble factors mediating the suppressive effect were composed of IL-10, transforming growth factor- and the hepatocyte growth factor.^{8, 11} However, as with most analyses of MSCs, these immune properties have been observed exclusively in cultured cells.

An immunosuppressive effect of MSCs in vivo has been shown in a baboon model, in which an infusion of ex vivo-expanded matched donor or third-party MSCs delayed the time of rejection of a histo-incompatible skin graft.¹² The immunosuppressive function of MSC grafts in human beings, as a corollary to the immunosuppressive effect of MSCs in vitro and in preclinical animal models, suggests that MSCs may be useful for preventing and treating GVHD in allo-SCT.^{13, 14} Recently, MSCs have been used successfully to treat a 9-year-old boy with severe treatment-resistant acute GVHD.¹⁵ However, the major focus on GVHD protection and the immunosuppression of MSCs has thus far been dominated by in vitro studies. Therefore, this study examined the effects of MSCs expanded ex vivo from the bone marrow of the donor type on the occurrence of GVHD in a well-established murine allo-SCT model of human GVHD. In this study, the primary MSCs but not genetically modified MSCs expressing IL-10 failed to protect against GVHD in this experimental allo-SCT model.

Materials and methods

Mice

Female C57BL/6 (B6, H-2^b CD45.2⁺), B6.Ly-5a (CD45.1⁺) and B6D2F1 (H-2^b/d CD45.2⁺) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The age of the mice ranged from 8 to 12 weeks. The mice were housed in sterilized microisolator cages and received filtered water and normal chow or autoclaved hyperchlorinated drinking water for the first 3 weeks after the allo-SCT.

Experimental allo-SCT and assessment of acute GVHD

The mice underwent transplantation according to standard protocol described previously.^{16, 17} Briefly, the recipients received a single dose of 1100 cGy total body irradiation (TBI; cesium Cs 137 [¹³⁷Cs] source). T cell-depleted bone marrow (BM) cells (10 10⁶) and 20 10⁶ splenocytes from the respective allogeneic or syngeneic donors were resuspended and injected intravenously into the recipient animals on day 0. The CD45.1⁺ B6Ly-5a mice were used as donors for the donor cell engraftment experiments. The survival was monitored daily, and the body weights and GVHD clinical scores of the recipients were measured weekly. The degree of systemic acute GVHD was assessed using a scoring system that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture and skin integrity. This scoring system is more accurate than just weight loss alone, as described previously.¹⁸ At the time of analysis, the mice from coded cages were evaluated and graded from 0 to 2 for each of the criteria. A clinical index was subsequently generated by adding the scores of the five criteria (maximum index, 10).

Isolation, proliferation and transfection of MSCs

The BM cells, which were collected by flushing the femurs and tibias with the medium, were cultivated in 75 cm² tissue culture flasks at a concentration of 1 10⁶ cells/ml using complete Dulbecco modified Eagle medium (WelGENE Inc., Deagu, South Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS, WelGENE Inc.), 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (Gibco BRL, Gaithersburg, MA, USA). No cytokines were added at any stage of the experiment. The cultures were incubated at 37°C in a 5% CO₂ atmosphere. After 72 h, the non-adherent cells were removed. When the cells had reached 70–80% confluence, the adherent cells were trypsinized (0.05% trypsin at 37°C for 5 min), harvested and expanded in larger flasks. A homogenous cell population was obtained after culturing for 3–5 weeks. The MSCs were maintained in culture for no more than 15 in vitro passages.

The IL-10 gene was inserted into a murine stem cell virus-based retroviral vector containing the internal ribosomal entry site and green fluorescent protein (GFP) (MIG). The MIG vectors containing the IL-10 gene were transfected into 293T cells using a lipofectin reagent (Invitrogen, Carlsbad, CA, USA). The cell-free supernatants from the 293T cells were collected and centrifuged to concentrate the viruses. The appropriate titer was 1.12 10⁷ IFU/ml. After drug selection with G418 (Sigma Chemical Co., St Louis, MO, USA), the cell-free supernatants were incubated with 1 10⁵ of the primary MSCs in the presence of 8 g/ml polybrene for infection with 50 multiplicity of infection. The most highly GFP⁺ MSCs were isolated using a fluorescence-activated cell sorter (FACS; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and subsequently expanded.

In vivo administration of MSCs or IL-10

MSCs (1 or 2 10⁶ per mouse) cultivated from the B6 mice were injected into the recipient animals intravenously on either day +1 or days +1, +3 and +5. Recombinant human (rh) IL-10, which is active in mice because of species cross-reactivity, was purchased from Preprotech (Seoul, Korea) and was reconstituted in phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA). Mice were injected intraperitoneally with rhIL-10 (3 or 30 g/day per mouse) on day +1.

Flow cytometry analysis

All the antibodies were purchased from PharMingen (San Diego, CA, USA). The procedure was performed as described elsewhere.¹⁹ Briefly, the cells were first incubated with mAb 2.4G2 for 15 min at 4°C and then with the relevant fluorescein isothiocyanate- or phycoerythrin-conjugated mAb for 30 min at 4°C. Finally, the cells were washed twice with PBS/0.2% bovine serum albumin and fixed with PBS/1% paraformaldehyde. Flow cytometry was performed using a FACSVantage SE cell sorter (Becton Dickinson). The identity of the MSCs was confirmed using the immunophenotype criteria, based on the expression of Sca1+ and the absence of hematopoietic (with anti-CD45, -CD11c and -CD117 antibodies) or endothelial cell (with anti-flk1 antibodies) markers. The proportion of CD45⁺ cells in the MSC preparations used in the various experiments never exceeded 2%. Double staining for CD45.1 and CD3⁺ was used to evaluate the expansion of donor T cells after allo-SCT.

Mixed lymphocyte reaction

The splenocytes were isolated from a mouse spleen by disaggregation into 10 ml RPMI 1640 medium (WelGENE Inc.). The cell count and viability were assessed by trypan blue dye exclusion. The stimulator splenocytes from the B6D2F1 mice were treated with 50 g/ml mitomycin C (Sigma Aldrich Co., MO, USA) for 45 min at 37°C, followed by five extensive washes with FBS-containing RPMI 1640 medium. The responder splenocytes from the B6 mice and the stimulator splenocytes from the B6D2F1 mice were resuspended in RPMI 1640 containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Gibco BRL), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 5 10^-5 M 2-mercaptoethanol (Invitrogen). Each responder and stimulator cell population was seeded in triplicate in 96-well round-bottom plates (BD Falcon, Bedford, MA, USA) at a concentration of 10⁵ cells/100 l/well. The MSCs (10⁵ cells) were added to the mixed lymphocyte reaction (MLR) to obtain a final volume of 300 l. In the mitogen proliferative assays, the responder splenocytes were incubated with 5 g/ml concanavalin A (ConA; Sigma Aldrich Co.).

Cytokine enzyme-linked immunosorbent assay

The amount of the cytokines (IFN-, TNF- and IL-10) in the culture supernatant and recipient mice was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (R&D Systems, Minneapolis, MN, USA). The samples and standards were run in duplicate. The absorbance at 450 nm was measured using a microplate spectrophotometer, Benchmark Plus (Bio-Rad, Richmond, CA, USA).

Statistical analysis

A Mann–Whitney U-test was used for the statistical analysis of the cytokine data, clinical GVHD scores and the number of cells, whereas the Wilcoxon rank test was used to analyze the survival data. A P-value<0.05 was considered significant.

Results

Isolation, in vitro expansion and phenotypic characterization of MSCs

MSCs were isolated from BM cells of C57BL/6 mice by plastic adherence in long-term culture. BM cells were initially cultured at a concentration of 10⁶ cells/ml. Each time cells reached confluence, they were detached and replated in culture. As shown in Figure 1a, the isolation of GFP⁺ MSCs containing either the MIG vector alone or IL-10 gene was performed using a fluorescence-activated cell sorter. We observed that cell expansion was markedly accelerated on both the IL-10-transduced MSCs (IL-10 MSCs) and the MIG vector-transduced MSCs (MIG MSCs). Both IL-10 and MIG MSCs were negative for CD45, CD11c, c-kit and flk1. In case of Sca1, most of MSCs from both types expressed this marker (Figure 1b).

Figure 1.

Comparative immunophenotype of MIG and IL-10 MSCs. (a) FACS sorting was performed based on GFP expression. (b) MIG or IL-10 MSCs were stained with surface antibodies and analyzed by FACS. The figure shows representative flow cytometry analyses.

Full figure and legend (68K)

Administration of donor-type primary MSCs did not ameliorate the severity of acute GVHD after experimental allo-SCT

It has been previously shown that an injection of MSCs can have immunosuppressive effects.²⁰ Therefore, this study examined whether or not primary MSCs can prevent or reduce the extent of acute GVHD after MHC class I- and II- disparate allo-SCT (B6, H-2^b B6D2F1, H-2^b/d). The dose of MSCs was first tested to determine a potential dose–response effect. The recipient B6D2F1 underwent transplantation with BM and splenocytes from allogeneic B6 or syngeneic donors, respectively, as described in 'Materials and methods'. The primary MSCs (1 or 2 10⁶ per mouse) cultivated from the B6 mice were injected into the recipient animals intravenously on day +1, whereas the control mice received identical injections of the diluent. The time of injection, day +1 vs days +1, +3 and +5 was then investigated to determine if it influences the severity of acute GVHD. As shown in Figure 2, either 1 or 2 10⁶ MSCs on day +1 did not have any beneficial effects in the recipients with acute GVHD. The lack of a GVHD protective effect on the primary MSCs was shown by the survival (Figure 2a) and clinical GVHD scores (Figure 2b). At lower doses (5 10⁵), a similar effect was observed. Animals that underwent syngeneic (syn-) SCT showed 100% survival, which ruled out any nonspecific toxicity of the conditioning. The GVHD scores for syn-SCT gradually returned to the baseline by week +2. No improvement in survival or acute GVHD severity was observed when the time of injection was changed from day +1 to days +1, +3 and +5 (data not shown). All the recipient animals displayed complete donor hematopoietic chimerism, as determined by FACS analysis (data not shown). This suggests that an early injection of MSCs after a transplant does not have any beneficial effect in this murine GVHD model.

Figure 2.

Effect of primary MSCs on survival (a) and GVHD score (b) in experimental acute GVHD induced across MHC class I and II. B6D2F1 mice received 1100 cGy TBI and underwent transplantation with 10 10⁶ BM cells and 20 10⁶ splenocytes from B6 or syngeneic F1 donors (Syn, , n=6). Either 1 10⁶ (MSC1, , n=10) or 2 10⁶ (MSC2, , n=10) culture-expanded MSCs from donor-type BM were injected on day +1. Controls (Con, , n=10) received the diluent on day +1. Survival and clinical GVHD scores were evaluated as detailed in 'Materials and methods'. Data from two similar experiments are combined. vs or , P>0.05.

Full figure and legend (20K)

Immunosuppressive function of IL-10-transduced MSCs

The efficacy of the viral form of IL-10 has already been shown to be a possible candidate for the self-renewal of hematopoietic cells²¹ and the treatment of arthritis.²² Therefore, the level of IL-10 secreted by MSCs in the presence or the absence of IL-10 gene transfection was measured. As shown in Figure 3a, the IL-10 concentration was significantly higher (up to 20-fold higher) in the fresh supernatants derived from the 2 days culture of the IL-10 MSCs than in the MIG MSCs. The abundant secretion of IL-10 from the IL-10 MSCs was maintained according to the culture time and the number of MSCs (Figure 3b).

Figure 3.

(a) Levels of IL-10 in the supernatant of MSCs in the presence or the absence of IL-10 gene transfections. Supernatants from 4 10⁴ MSCs were collected at 48 h and assayed by ELISA for IL-10 as described in 'Materials and methods'. Each graph represents one of three similar experiments. ^†Denotes 10 in vitro passages and ^‡11. MIG vector vs IL-10, ^*P<0.01. Error bars represent standard error. (b) Production of IL-10 in the supernatant of MSCs (10 in vitro passages) according to incubation time and MSC number. MIG MSCs vs IL-10 MSCs, ^*P<0.01. IL-10 MSCs 4 10⁴ vs 8 10⁴, ^**P<0.05. Error bars represent standard error.

Full figure and legend (92K)

The immunological properties of the IL-10 MSCs were compared with the MIG MSCs by a proliferative assay, using the splenocytes of the B6 mice as the responding cells and ConA as the mitogen. Compared with the diluent alone (control), the ConA-induced proliferation of the responder B6 splenocytes were sharply inhibited by the addition of the IL-10 or MIG MSCs (Figure 4a). Using a MLR, the MIG and IL-10 MSCs suppressed the proliferation of responder B6 splenocytes that had been elicited by allogeneic splenocytes from the B6D2F1 (Figure 4b). The addition of the IL-10 MSCs inhibited the proliferative response of allogeneic responder splenocytes more efficiently compared with the addition of MIG MSCs, suggesting more potent immunosuppression. These data indicate that IL-10 MSCs inhibited in vitro T cell receptor (TCR)-dependent proliferation more effectively than the MIG MSCs. However, the inhibition of TCR – independent T cell proliferation by the addition of either the IL-10 or MIG-MSCs was similar.

Figure 4.

(a) Responding B6 splenocytes (10⁵ cells) were incubated for 2 days with either 5 g/ml concanavalin A (ConA) with 1 10⁵ IL-10 MSCs (IL-10), MIG MSCs (MIG) or diluent alone (Control). T cell receptor-independent (ConA) T cell proliferation was inhibited by both IL-10 and MIG MSCs compared to controls (^**P<0.01). (b) Responding B6 splenocytes (10⁵ cells) were incubated for 4 days with either mitomycin-treated B6 (Syngeneic) or B6D2F1 in the presence of 1 10⁵ IL-10 MSCs (IL-10), MIG MSCs (MIG) or diluent alone (Control). Addition of IL-10 MSCs inhibited T cell receptor-dependent (allogeneic) T cell proliferative response more potently compared to MIG MSCs (^*P<0.05) as well as the controls (^**P<0.01). Each graph represents one of the three similar experiments.

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Effects of systemic injection of IL-10 MSCs on the severity of acute GVHD

As the donor-type primary MSCs did not display any immunosuppression in the MHC-mismatched allo-SCT model, this study investigated whether or not the use of genetically modified MSCs, which expressed high amount of IL-10, could attenuate the severity of acute GVHD. The recipient B6D2F1 mice underwent transplantation, as described in 'Materials and methods'. Either 2 10⁶ IL-10 or MIG MSCs were injected in the tail vein of the mice on day +1. The controls received an i.v. injection of an equal volume of PBS at the same time. This dose and time of the cell injection were chosen based on the fact that the activation of host dendritic cells and donor T cells occur extremely early in transplant recipients after irradiation and stem cell infusion,²³ while still permitting significant IL-10 secretion. The recipient survival and acute GVHD score were monitored as a measure of the severity of acute GVHD by at least the 50th day after the transplantation. As shown in Figure 5a, compared with the injection of MIG MSCs or the controls, the injection of the IL-10 MSCs significantly delayed the acute GVHD-related deaths at day 50 after the MHC class I- and II-disparate SCT (percent survival, 0, 10 vs 70%, P=0.0004 or 0.0064, respectively). In stark contrast, the injection of the MIG MSCs did not decrease the severity of the acute GVHD-related mortality compared with allogeneic controls (percent survival, 0 vs 10%, P=0.064). The reduction in mortality was confirmed by the semi-quantitative GVHD score measured on from day +7 to day +50 (P<0.05, Figure 5b). The production of cytokines such as IFN- and TNF- and the expansion of the donor T cells have been implicated in GVHD-related mortality.^{1, 24} Therefore, serum levels of IFN- and TNF- were measured on day +7 after the allo-SCT. As shown in Figure 6, the level of IFN- was markedly lower in the recipients of the IL-10 MSCs than in either those of the MIG MSCs or controls, whereas the levels of TNF- of each group were not different. The reduced GVHD severity was associated with lower serum levels of the pro-inflammatory cytokine, IFN-, on day +7 (IL-10 MSCs vs MIG MSCs; 297116 vs 68187 pg/ml, P=0.015). The level of donor CD3⁺ T cell expansion on day +7 after the transplant was similar in the three allogeneic groups (data not shown). This shows that the MSCs engineered to express an anti-inflammatory cytokine, IL-10, had beneficial effects on acute GVHD.

Figure 5.

Effect of IL-10 MSCs on survival (a) and GVHD score (b) in experimental acute GVHD induced across MHC class I and II. B6D2F1 mice received 1100 cGy TBI and underwent transplantation with 10 10⁶ BM cells and 20 10⁶ splenocytes from B6 or syngeneic F1 donors (Syn, , n=6). Allogeneic recipients were injected on day +1 with either 2 10⁶ IL-10 MSCs (, n=10) or 2 10⁶ MIG MSCs (, n=10) of donor type were injected on day +1. Allogeneic controls (Con, , n=10) received the same amount of diluent on day +1. Survival and clinical GVHD scores were evaluated as detailed in 'Materials and methods'. Data from two similar experiments are combined. The difference in the survival rate was evaluated with the Wilcoxon rank test. vs or , P=0.0004 or P=0.0064, respectively. Clinical severity was reduced with the injection of IL-10 MSCs. ^*P<0.05.

Full figure and legend (22K)

Figure 6.

Effect of IL-10 MSCs on serum inflammatory cytokines after allo-SCT. B6D2F1 mice underwent transplantation with BM and splenocytes from syngeneic or allogeneic donors and were injected with IL-10 MSCs, MIG MSCs or the diluent as in Figure 4a. Sera from the recipient animals (n=3–4/group) were obtained on day +7 after allo-SCT and were analyzed as described in 'Materials and methods'. Serum levels of IFN- (a) were significantly decreased in the IL-10 MSC-injected allogeneic recipients compared to those injected with MIG MSCs or the diluent (^*P<0.05). There were no significant differences in the serum levels of TNF- (b) among allogeneic recipients.

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Exogenous administration of IL-10 failed to decrease GVHD-induced mortality

To understand the reason why IL-10 MSCs suppressed GVHD mortality, we chose rhIL-10 intraperitoneal injection schedule in doses ranging from 3 to 30 g/injection on day +1. Two experiments were performed under identical conditions, and survival data have been pooled. As shown in Figure 7a, recipients of the rhIL-10 injections experienced a similar survival rate as compared with those of PBS injection. The fact that rhIL-10 failed to decrease GVHD-induced mortality is further supported by the similar mean weights between IL-10-treated mice and controls, which were noted at multiple time periods during the first 3–5 weeks post-allo-SCT (Figure 7b).

Figure 7.

Effect of exogenous IL-10 administration on survival (a) and body weight loss (b) in experimental acute GVHD induced across MHC class I and II. B6D2F1 mice received 1100 cGy TBI and underwent transplantation with 10 10⁶ BM cells and 20 10⁶ splenocytes from B6 or syngeneic F1 donors (Syn, , n=6). Either 3 g (3 IL-10, , n=10) or 30 g (30 IL-10, , n=10) rhIL-10 was injected on day +1. Controls (Con, , n=10) received the diluent on day +1. Survival and body weight loss were evaluated as detailed in 'Materials and methods'. Data from two similar experiments are combined. vs or , P>0.05.

Full figure and legend (21K)

Discussion

The immunosuppressive properties of MSCs have been established,²⁵ and recently, the treatment of GVHD with MSCs has been reported,^{14, 15} which suggests that MSCs can inhibit undesirable immune responses. This study examined whether or not murine MSCs of the donor type might have protective value in a well-established mouse model of human GVHD. Unexpectedly, the early administration of the primary MSCs after the transplant failed to show a GVHD protection effect. The MSCs failed to show any immunosuppression in this experimental GVHD model and have been shown to induce an immunosuppressive microenvironment through the production of IL-10. Therefore, this study investigated whether or not the use of IL-10-transduced MSCs has a GVHD protection effect. Genetically modified MSCs, expressing a high amount of IL-10 attenuated the severity of acute GVHD more efficiently, which was paralleled by the significant suppression of IFN- production, supporting the profound inhibition of the alloreactive T cell response. On the contrary, exogenous injection of IL-10 did not attenuate the severity of acute GVHD. The administration of donor-derived BM stromal cells in combination with the supplement of cytokines may be more desirable for the complete regulation of the occurrence of lethal GVHD after allo-SCT.

The characterization of the cytokines produced by MSCs is still provisional and is hindered by the lack of standardization in the isolation and culture conditions. Some of these cytokines provide critical cell–cell interactions and promote hematopoietic stem cell differentiation. However, caution should be exercised before interpreting these findings.²⁵ IL-10 has a well-documented role in the regulation of T cells as well as in the promotion of a 'regulatory' or suppressor phenotype. Human MSCs constitutively produce IL-10,²⁶ whereas Rasmusson et al.²⁷ and Beyth et al.²⁸ only detected IL-10 in co-culture experiments. MSCs are easily transfected by a variety of vectors, and can be envisioned as vehicles for either short-or long-term gene transfer,²⁹ which could facilitate the therapeutic effect of MSCs. Although the primary MSCs with the vector alone showed some in vitro immunosuppressive potential, no beneficial effect on the acute GVHD was observed when they were injected early into allo-SCT recipients. It is unlikely that supplying MSCs alone completely alleviated the severity of acute GVHD after allo-SCT. Therefore, the use of genetically engineered, culture-expanded BM-derived MSCs, as delivery agents for an anti-inflammatory cytokine, might be another strategy. This is supported by the observation of the specific homing of intravenous administrated MSCs, engineered to produce IFN-, to tumors with subsequent regression of a tumor in a xenogeneic mouse model.³⁰ In our study, the IL-10 MSCs suppressed the in vitro proliferative activity of allogeneic splenocytes and produced IL-10 more efficiently than the MIG MSCs, which was extended to a preclinical murine GVHD model. A potent protective effect against acute GVHD was observed with a single dose of the genetically modified MSCs administered intravenously. The lower GVHD score demonstrated the clinical efficacy of this treatment, which was consistent with the severity of acute GVHD. These results are consistent with the effect being observed during the early inflammatory phase of acute GVHD, which supports the possibility that IL-10 MSCs can affect the generation of alloreactive effector T cells when the donor T cells in the allograft are exposed to the host antigens.

The mechanism involved in the in vivo tolerance induced by IL-10 MSCs is unclear, although the role of soluble factors has been suggested.^{8, 10} IL-10 is an ideal candidate cytokine for inhibiting the acute GVHD response because it is a potent inhibitor of both the proliferative and cytolytic responses of murine T cells³¹ and inhibits the production of inflammatory cytokines, which are derived from macrophages that are activated during GVHD.³² This study showed that the reduced GVHD mortality by an early injection of IL-10 MSCs could be explained by its potential interactions with and the down regulation of IFN- production. Allen et al.³³ reported that the splenic IL-10 mRNA levels are markedly elevated in the B6D2F1 recipients of a B6 parental graft. These mice develop acute suppressive GVHD that is associated with high levels of IFN- and no elevation in the IL-4 levels, suggesting an interaction between these cytokines. The role of IFN- in regulating GVHD is unclear. A sustained high level of IFN- production can result in more severe GVHD in some models.^{16, 34} More recently, an enzyme-linked immunospot assay, which reflects ongoing immune status in vivo, showed that the level of spot-forming cells (SFCs) for IFN- were significantly higher in patients with high-grade acute GVHD, suggesting a correlation between the number of IFN- SFCs and clinically significant acute GVHD.³⁵

Although the immunosuppressive properties of IL-10 are well known, IL-10 also has immunostimulatory effects. IL-10 increases MHC II expression on small resting B cells and drives the proliferation of activated B cells.³⁶ IL-10 augments the in vitro proliferative responses of IL-2- and IL-4-activated T cells, and increases the precursor frequency and function of the IL-2-stimulated murine cytolytic T cell precursor generated from mitogen-stimulated CD8⁺ splenocytes.^{37, 38} Consistent with the immunostimulatory effects of IL-10, exogenous IL-10 injections at high but nontoxic doses accelerated the GVHD-induced lethality and increased the host-mediated resistance to allogeneic engraftment.³⁹ Paradoxically, low doses of IL-10 released by donor T cells protected the mice against GVHD lethality.⁴⁰ In our experiments, treatment with exogenous IL-10 failed to attenuate the severity of acute GVHD at the dose of 3–30 g/mouse on day +1, whereas IL-10 MSCs on day+1 showed GVHD protection. This suggests that the amount of bioavailable IL-10 present at the critical stages of GVHD development may markedly influence the lethality of GVHD.

The relapse of malignancies after a transplant remains a significant problem in allo-SCT, and a further dose escalation of the conditioning regimens is limited by their toxicity to nonhematopoietic organs such as the lungs and digestive system.⁴¹ Unfortunately, the GVHD and a graft-versus-tumor (GVT) effect are tightly linked, as demonstrated by the inverse correlation between the leukemia relapse rates and the severity of GVHD.⁴² The prevention of GVHD by T cell depletion or nonspecific immune suppression is associated with the increased risk of relapse after allo-SCT.⁴³ Allogeneic CD8⁺ T cells induce more severe systemic GVHD but weaker GVT/antihost lymphohematopoietic reactions if they do not produce IFN-, suggesting that the production of IFN- by donor cells plays an important role in the induction of GVT reactions.⁴⁴ One concern with use of IL-10 MSCs is the potential to increase the incidence of relapse as a result of severe immunosuppression. Further experiments aimed at determining if IL-10 MSCs can preserve the GVT effect conferred by the donor T cells after allo-SCT in this animal model are currently underway.

In summary, several clinical trials of a MSC infusion have been reported or initiated.^{13, 14, 15} The experimental basis for these concepts is somewhat limited. A limited number of human studies have highlighted the potential of human MSCs to suppress GVHD. Despite these findings, this study found that an intravenous injection of donor-type, ex-vivo cultured MSCs at the early time of GVHD induction in a well-established murine model of human GVHD did not attenuate the severity of acute GVHD. The effect of a single dose of IL-10 MSCs intravenously was superior to that of the culture-expanded primary MSCs having the vector alone. The optimization of MSC therapeutic applications will require innovative approaches that extend beyond the relatively simple expansion and transplantation techniques currently used. Clinical strategies involving the use of MSCs engineered with cytokines are likely to be more powerful in overcoming GVHD provided the effects against GVT are not diminished.

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Acknowledgements

This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0420130-2). A grant of high-performance cell therapy R&D project (0405-DB01-0104-0006) by the Ministry of Health & Welfare partly supported this work.