CCR7 Expressing Mesenchymal Stem Cells Potently Inhibit Graft-versus-Host Disease by Spoiling the Fourth Supplemental Billingham’s Tenet

The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham’s tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.


Introduction
Graft-versus-host disease (GvHD), a representative of T cell-mediated immune responses, remains a significant cause of morbidity and mortality in patients undergoing bone marrow transplantation. Billingham's tenets reflect the three basic principles in the development of GvHD [1]. Additionally, some investigations highlighted that the effector cells migrating to the target tissues is important for the development of GvHD. FTY720 inhibited GvHD lethality by preventing lymphocyte egress from Secondary lymphoid organs (SLOs) to peripheral organs [2][3][4]. Corticosteroids, the first-line therapy of GvHD, make lymphocytes trafficking into bone marrow, but away from lymph nodes and inflammatory organs. All these proved the critical role of the lymphocyte homing requirement in the GvHD development, which was proposed as a corollary to Billingham's criteria [5]. Meanwhile, this provided chances to modulate GvHD by controlling lymphocyte trafficking [6,7].
Mesenchymal stromal cells (MSCs) are multipotent non-hematopoietic progenitor cells of stromal origin that can be isolated from the bone marrow or other tissues (adipose tissue, cord blood) [8][9][10][11]. MSCs have potent immunomodulatory effects. When cultivated with dendritic cells (DCs), T-lymphocytes and NK cells, they can shift them to the anti-inflammatory phenotypes. Some soluble factors participate in this processes, such as IL10, nitric oxide (NO), indoleamine 2,3-dioxygenase, prostaglandin E2, etc [12][13][14]. Therefore, MSCs have been employed to treat various immune disorders in animal models and clinical settings.
SLOs, including spleen (SP), lymphoid nodes (LN), mesenteric lymphoid nodes (MLN), Peyer's Patches (PP), etc, are'hubs'of immune surveillance [15,16]. Our previous study showed that CCR7 guide the migration of MSCs to SLOs, separate GvHD from GvL effect [17]. In this study, we further demonstrated that the inducible immunomodulatory activity in vitro of MSCs/CCR7 is depending on the NO production. Transfused MSCs/CCR7 relocate at the appropriate T cellrich zones within SLOs and inhibit GvHD lethality through spoiling the fourth supplemental Billingham's tenet.

Ethics statement
This study was carried out in strict accordance with the recommendations in the national guidelines for the use of animals in scientific research ''Regulations for the Administration of Affairs Concerning Experimental Animals''. The protocol was also approved by the Animal Care and Use Committee of Beijing Institute of Basic Medical Sciences (Permit Number BMS-1104139), and all efforts were made to minimize suffering.

Mice
Inbred BALB/c (H-2d) and C57BL/6 (H-2b) male mice were purchased from the Laboratory Animal Center, Academy of Military Medical Sciences. Animals were maintained under specific pathogen-free conditions and all animal experiments were performed in accordance with the Academy of Military Medical Sciences Guide for Laboratory Animals.

MSCs culture
Primary MSCs were isolated from murine compact bone and culture-expanded as described in our previous report [18], and grown in minimal essential medium (MEM, Gibco) with 4 mM L-glutamine, 100 U /ml penicillin, 100 U /ml streptomycin and 10% fetal bovine serum (FCS) in a humidified atmosphere of 5% CO 2 at 37˚C.
Inducible nitric oxide synthase (iNOS) detection [19] MSCs, MSCs/eGFP and MSCs/CCR7 were planted on the microscope cover glasses (NEST) in the 24-well plate overnight and treated with IFNc plus TNFa (2 ng/ml each) for another 72 hours. Then cells were collected for immunofluorescence detection using the polyclonal iNOS antibody, followed by PE goat anti-mouse IgG (BD Transduction Laboratories). Confocal images were collected by the Zeiss LSM510 Meta and were acquired using a LSM image browser.

Detection of NO
NO in culture supernatants was detected using a modified Griess reagent (Sigma-Aldrich). Briefly, all NO 3 is converted into NO 2 by nitrate reductase, and total NO 2 detected by the Griess reaction. NaNO 2 served as a standard.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining CD3 + T cells selected with CD3e MicroBead Kits (MiltenyiBiotec) were labeled with 5 mM CFSE (Invitrogen) for 8 min at 37˚C with gentle vortex every 2 min. The labeling was terminated by adding equal volume of FCS. After washing, cells were cultured with different dose of MSCs/eGFP or MSCs/CCR7 in the presence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 mg/ml ionomycin (ION, Sigma) for 48 hours. Cell division, as evidenced by reduction of fluorescence intensity, was analyzed by FCM.

In vivo distribution of transplanted MSCs
In order to detect the specific anatomic distribution within SLOs of transplanted MSCs/eGFP or MSCs/CCR7, cells (5610 5 ) were injected into the lateral tail vein of GvHD mice in a total volume of 0.2 ml PBS. Five days later, samples of the SP, LN from the recipients were collected for cryosection. For immunofluorescent staining, slides were fixed in cold acetone for 10 minutes, and then washed for 10 minutes in PBS. Slides were stained with a PE-conjugated anti-mouse CD3, B220 or CD11c antibody (BioLegend). The sections were counter-stained with 1.0 mg/ml 49, 6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) in PBS for 20 minutes at room temperature in the dark. Fluorescent cells on sections were visualized under Olympus CK2 fluorescence microscope.

Murine GvHD model
Bone marrow cells (BMC) were obtained from BALB/c mice followed by red blood cell lysis. Splenic mononuleocytes (SPMNC) were isolated by Ficoll gradient centrifugation from Balb/C mice. In the GvHD group, 1610 7 BMC and 2610 7 SPMNC in a total volume of 0.2 ml PBS were injected into lethally irradiated (9Gy) C57BL/6 mice through lateral tail vein. MSCs/eGFP or MSCs/CCR7 (5610 5 ) were co-injected into GvHD mice, which were defined as GvHD+MSCs/ eGFP and GvHD+MSCs/CCR7 groups of mice respectively.
The condition of the animals was monitored daily. The degrees of systemic acute GvHD were assigned a score from 0 to 2 for each of 5 GvHD parameters: weight loss, activity, fur-ruffling, kyphosis and skin lesions. Scores ranged from 0 (minimum) to 10 (maximum) [20]. Animals that developed clinical symptoms of GvHD (40% weight loss, hunched posture, fur loss, reduced mobility, tachypnea) were sacrificed in their homecage by CO 2 inhalation to effect and an end point of survival was recorded for all GvHD mice. At different time points after infusion, mice were sacrificed by CO 2 inhalation, and specimens from at least three recipient mice per group were collected for experiments.

T cell proliferation assay
T cells in spleens of the three groups of mice were isolated with pan-T isolation kit (MiltenyiBiotec) according to the manufacturer's instructions. T lymphocytes (5610 5 /well) were planted in the 96-wells plate in RPMI 1640 supplemented with 20% FBS, and 100 U/mL IL-2 (Peprotech). The total volume was 200 ml per well. Plate was maintained at 37˚C for 5 days and pulsed with 5 mCi of 3 H-thymidine deoxyribonucleoside/ml for an additional 18 hours. Cells were then harvested onto glass fiber filters and radioactivity was measured on a Wallac Microbeta Trilux 1450-02P.

Cytotoxicity assay
Cytotoxicity assay was performed with lactate dehydrogenase cytotoxicity detection kit (BioVision). T cells in spleens were isolated with pan-T isolation kit (MiltenyiBiotec). Cells were then incubated with 4610 4 EL4 leukemia cells for 12 hours on the 96-well plates. The wells of low control contain 4610 4 EL4 cells only in medium and the wells of high control contain 4610 4 EL4 cells in 1% Triton X-100. The absorbance at 450 nm, which represents the values of LDH release, was measured in an enzyme-linked immunosorbent assay (ELISA) reader. The cytotoxicity (%)5(Test Sample-low control)/(High control-low control)6100.

Statistical analysis
The Kaplan-Meier product-limit method was used to calculate survival curve. Differences between groups in survival studies were determined using log-rank statistics. For all other data, a Student's t test was used to analyze differences between groups, and results were considered significant if the P value was less than or equal to 0.05.

CCR7 expression in MSCs
CCR7 can guide various types of immune cells to SLOs [21][22][23][24][25][26]. Both hBM-MSCs and hUC-MSCs at passage 5 expressed traceable CCR7 mRNA detected by RT-PCR and FCM assay (Fig. 1A, 1B). Similarly, CCR7 was negligible in murine MSCs (Fig. 1C, 1D). Then, we transduced MSCs from C57BL/6 mouse strain with lentivirus carrying CCR7 gene. This group of cells was defined as MSCs/CCR7 in the following experiments. MSCs transduced with the control lentivirus were also referred as negative control (MSCs/eGFP). CCR7 can be successfully detected only in MSCs/CCR7at mRNA level detected by RT-PCR assay, and at cell surface protein level examined by FCM. The eGFP gene and encoded protein could be both detected in the MSCs/eGFP and MSCs/CCR7 (Fig. 1E, F).

iNOS expression in MSCs/CCR7
Previous investigations revealed that NO is solely generated by iNOS in murine MSCs and plays a critical role for their immunoregulatory function exerting [19,27,28]. We examined the iNOS level in MSCs, MSCs/eGFP and MSCs/CCR7. Contrasted to the cells without stimulation, iNOS mRNA increased nearly 100 folds in the three types of MSCs provoked by IFNc plus TNFa for 72 hours ( Fig. 2A). In situ immunofluorescence staining results confirmed that iNOS expression could be elicited by inflammatory cytokine stimulation (Fig. 2B). Though there were little dose without stimulation, the NO levels in the supernatant of three groups of MSCs were dramatically increased under inflammatory stimulation in a time dependent way (Fig. 2C).

Immunomodulatory capacity of MSCs/CCR7 in vitro
Next, we examined whether CCR7 carrying MSCs still have immunomodulatory capacity in vitro. CFSE labeled CD3 + T cells in the presence of PMA plus ION were cultured for 48 hours. The CFSE on the T cells were significantly diluted with proliferation. Co-culture of MSCs/eGFP or MSCs/CCR7 inhibited T cell proliferation in a dose dependant manner (Fig. 3A). NG-monomethyl-L-arginine acetate salt (L-NMMA, 2 mM),the iNOS inhibitor, was added to the T cell proliferation assay system (MSCs:T cells51:10). As expected, L-NMMA treatment reinstated the T-cell proliferation in the co-culture assay (Fig. 3B). These results indicated that MSCs/CCR7 have similar immunomodulatory function in a NOdependent manner as MSCs/eGFP.

SLOs are constructed with T cells, B cells, antigen-presenting cells (APCs), stromal cells and vascular supplies. CCR7programmed
MSCs migrate to and concentrate in SLOs after infusion (Fig. 4A). However, the precise anatomic sites in SLOs that the transfused MSCs/CCR7 found are crucial for their in vivo function exerting. Interestingly, our immunofluorescent staining results revealed that MSCs/CCR7 relocated at the right position of T cell-rich zones (relocated withCD3 + T lymphocytes and CD11c + dentritic cells nearby, but far from B220 + B cell follicle, Fig. 4B-D). This set a foundation for MSCs/CCR7 exerting potent immunoregulatory activity on the T cell immune response.

MSCs/CCR7 Co-infusion potently inhibited the lethality of GvHD
Donor na ve T cells initially traffic to the recipient SLOs when GvHD occurs, then they undergo activation, expansion, and subsequent migration to peripheral target organs [29,30]. Since MSCs/CCR7 relocated at the right position of T cell-rich zones within SLOs, we next tested their in vivo activities in GvHD model, which is a representative T-cells response disease.
Co-injection of 5610 5 MSCs did not prolong the survival time of GvHD mice. Captivatingly, co-injection of 5610 5 MSCs/CCR7 significantly enhanced the survival of the recipients (Fig. 5A). Recipient animals were evaluated using a validated clinical GvHD scoring system. Comparing with the mice ofGvHD and GvHD+MSCs groups, mice of GvHD+MSCs/CCR7 group also had considerable alleviated scores (Fig. 5B-C). These results demonstrated that MSCs/CCR7-eGFP were more efficient in immunomodulation, presumably due to the pivotal relocation site they hold in SLOs.
Pathologic examination confirmed that MSCs/CCR7 co-infusion declined the lymphocyte infiltration in the liver, small intestine. Comparing to the obvious lymphocyte existence in the spleens of the MSCs/CCR7 treated mice, there was large area of cellular atrophy and necrosis in the spleens of GvHD and GvHD+MSCs groups of mice (Fig. 5D).

MSCs/CCR7 Co-infusion made the T cells in SLOs more naı¨ve like and thus control of T cells trafficking from SLOs to the target organs
We further investigate the mechanism of in vivo immunoregulatory effects of MSCs/CCR7. Since MSCs/CCR7 relocated at the right position of T cell-rich zones within SLOs after infusion, as expected, the proliferations of T cells in SP were significantly restrained by MSCs/CCR7 co-infusion (Fig. 6A). Moreover, their cytotoxicities were also significantly suppressed (Fig. 6B).
CD3 + T cells in SP were increased gradually as GvHD developing. Noteworthily, in contrast to the lower proliferation and lower cytotoxity of T cells, there were higher CD3 + T cells in SP of the MSCs/CCR7 co-infusion group of mice (Fig. 6C). Further examination revealed that the increased T cells were more na ve like (the increased proportion of CD62L + CCR7 + in CD3 + cells) (Fig. 6D). Similar results   were found in the LN, MLN and PP (data not shown). On the other hand, the CD3 + T cells infiltrated in the liver and small intestine declined (Fig. 6E). Tregs is a natural ''suppressor'' of the immune system. Our data showed that the proportion of Tregs remarkably increased in SP of the GvHD+MSCs/CCR7 group of mice (Fig. 6F). MSCs/CCR7 Inhibit GvHD by Control of T Cells Trafficking

Discussion
As the clinical GvHD-therapy result of MSCs is not as satisfactory as expected in multicenter phase III clinical studies [31][32][33][34]. The aim of our present study is to further investigate the mechanism of enhanced in vivo immunomodulatory of CCR7 carrying MSCs.
CCR7 can guide various types of cell to SLOs, where generate immune responses or induce tolerance. As one kind of professional APCs, DCs are the most potent initiator of in vivo immune responses [35][36][37]. Upon maturation, DCs upregulate CCR7 expression and migrate from the peripheral tissues to the T-cell regions of SLOs, followed by instigating T-cell activation [21][22][23][24][25]. Regulatory T cells (Tregs) are instrumental to induce and maintain tolerance in transplantation immune response [38][39][40]. Moreover, Tregs mature in the SLOs of the recipients. Tregs induce allo-tolerance by interacting with APCs and T cells, which process requires their proper homing to the lymphoid tissues. CCR7 expression is important not only for Treg homing to the draining LN, but also for optimal Tregs suppressive function exerting [25].
Since murine MSCs and human MSCs scarcely express CCR7 at the mRNA level and cell surface protein level [41][42][43][44][45], we introduce CCR7 gene into murine MSCs by lentivirus infection. To our excitement, CCR7 carrying MSCs can target migrate to and relocate at the appropriate T cell-rich zones within SLOs, which set a foundation for MSCs/CCR7 to shape T cell immune response in vivo. Owing to the pivotal relocation sites, MSCs/CCR7 at the same dosage displayed enhanced effect than normal MSCs in prolonging the survival and alleviating the clinical scores of GvHD mice (Fig. 5A-C).
T lymphocytes are classified into four different subsets. Expressing CD62L and CCR7, Na ve T and central memory T cells can home to SLOs. Activated by APCs, effector memory T and effector T cells lose the expression of CD62L and CCR7, emigrate from SLOs into the peripheral inflammatory tissues [46][47][48]. Our results showed that MSCs/CCR7 infusion dramatically made T cells in SLOs less proliferous and cytotoxic. MSCs/CCR7 made donor T lymphocyte in SLOs more 'na ve like' (the higher expression levels of CD62L and CCR7 molecules on the CD3 + T lymphocytes) (Fig. 6D). The increased na ve phenotype may give explanation to the increased donor T cells in SLOs and less lymphocytes infiltration in the peripheral target organs. Therefore, it was justifiable that MSCs/ CCR7 made the T cells maintain in SLOs (Fig. 6C), and decrease infiltration in GvHD target organs (Fig. 6E). These results mean MSCs/CCR7 infusion spoiling the fourth supplemental Billingham's tenets to inhibit GvHD development.
Consistent with the recent studies, the immunomodulatory effect of MSCs/ CCR7 was not innate, and it could be induced by inflammatory stimulation (Fig. 2). This may confer the inducible modulatory activity of MSCs/CCR7 in vivo. In the other words, acting as potent inflammation holder, MSCs/CCR7 might calm down the high immune process quickly and keep quiet in the normal physiological circumstance as well.
Tregs is a natural ''suppressor'' of the immune system [49,50]. It is generally recognized that Tregs treatment can reduce GvHD lethality [51,52]. In our study, MSCs/CCR7 infusion significantly increased the Tregs population, which joined forces in making the T cells more na ve like in SLOs.

Conclusion
Though CCR7-expressing MSCs exhibit equal modulatory activities as MSCs on T cell immune response in a NO-dependent manner in vitro, they can relocate at the appropriate T cell-rich zones within SLOs after infusion. MSCs/CCR7 co-infusion potently made the T cells in SLOs more na ve like, thus coerce T cells stay in SLOs from trafficking to the target organs. Through spoiling the fourth supplemental Billingham's tenets, MSCs/CCR7 potently inhibited the development of GvHD.
The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulation effect for clinical immune disease therapy.