| Background and Objection:Allo-hematopoetic stem cell transplantation (Allo-HSCT) is used for curing maglignant hematological diseases.50%～70%recipients have graft-versus-host disease (GVHD), which is a major cause of morbidity in up to50%of transplant recipients. The pathophysiology of GVHD is complicated and is still unclear. It has been considered that major histocompatibility complex (MHC) differences between donor and recipient induce donor T cell activation, Thl and Th2differentiation and cytokine cascade, then induce acute and chronic GVHD. Acute GVHD (aGVHD) can be summarized in a three-step process. In step Ⅰ, the intensive irradiation and (or) chemotherapy lead to the damage of host tissues, stimulating the cascade secretion of the pro-inflammatory cytokines tumor necrosis factor (TNFa) and interlukin-1(IL-1). These cytokines increase the expression of MHC antigens and activate antigen-presenting cells (APC). In step Ⅱ, donor T cells recongnize allogeneic antigen presented by APC and activate, then proliferate and differentiate into effectory cells, which is characterized by the predominance of Thl cells and the secretion of TNFa and interferon y (IFNγ) In step Ⅲ, activated macrophages, along with cytotoxic lymphocytse, secrete inflammatory cytokines such as TNFa that cause target cell apoptosis. Graft-versus-leukemia (GVL) effects are mediated predominantly by alloreactive T cells that also cause graft versus host disease (GVHD) in allo-HSCT recipients, which play an important role in prevention of leukemia relapse. Recipient dendritic cells (DC) and other APC induce the activation and differentiation of donor allreactive T cells. Critical cellular interactions in the pathogenesis of GVHD occur in secondary lymphoid organs such as mesenteric lymph nodes (MLN) that function as the meeting ground between host APC and donor T cells. After total body irradiation (TBI) or chemotherapy, increased proinflammatory cytokines such as IFNy augment expression of CCR7by DC and release of the CCR7ligands CCL19, CCL21in LN. CCR7expressed by DC and the CCR7ligands mediate the migration of recipient DC from tissue to draining LN, where they induce donor T cell expression of tissue-specific homing and chemokine receptors. In MLN, donor T cells interact with recipient CD103+DCs and up-regulate expression of gut-homing receptors, including a4β7and CCR9. In peripheral lymph nodes (PLN), donor T cells interact with recipient DC to up-regulate expression of skin-homing receptors, including E-ligand, P-ligand, CCR4and CCR10. Activated T cells subsequently migrate to epithelial tissues such as gut and skin to cause GVHD.In a recent report, we have showed that treatment with Fc receptor binding (FB) anti-CD3along with total body irradiation (TBI) before HSCT down-regualted expresison of gut and skin homing and chemokine receptors by donor T cells in MLN and PLN, respectively. As a result, donor T cells did not migrate into GVHD target organs (i.e gut and skin) where they could cause GVHD. Most of donor T cells were confined to lympho-hematopoietic tissues where malignant leukemia or lymphoma cells reside. Taken together, these results showed that pre-conditioning with FB anti-CD3before HSCT prevents GVHD while preserving GVL effects. We recently observed that CD103+DCs in MLN include both CCR7+and CCR7" subsets. DC in PLN are CD103-but also include CCR7+and CCR7" subsets. It has been proved by in vitro and in vivo experiments that only CCR7+DC not CCR7-DC can induce donor T cells expression of homing and chemokine receptors. In addition, we previously showed that CCR7+DC in MLN were depleted after FB anti-CD3conditioning. So we presume that depletion of CCR7+DC is associated with prevention of GVHD and preservation of GVL effects by trearment with FB anti-CD3before HSCT, but the mechanisms of depletion have not been defined.It has been reported that administration of FB anti-CD3activated T cells and triggered the release of interlukin-2(IL-2), TNFa, IFNy and interlukin-6(IL-6), thereby cansing "cytokine storm sydrome" both in rodents and human. An Fc receptor non-binding (FNB) anti-CD3mAb with the same viriable region as the FB anti-CD3(clone145-2C11), but with a murine IgG3constant region, dose not have the ability of binding to Fc receptor (FcR). Injection of FNB anti-CD3IgG3causes littile T cell activation or release of IL-2and TNFa, avoiding "cytokine storm syndrome". We previously observed that treatment with FNB anti-CD3IgG3indeed reduced "cytokine storm syndrome", but resluted in partial CCR7+DC depletion, and can not prevent GVHD effectively. These results indicate that released cytokines and anti-CD3FcR binding ability may be reqiured for FB anti-CD3mediated CCR7depletion and GVHD prevention. In current study, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and flow cytometry to detect CCR7+DC apoptosis. Meanwhile, we explored how FB anti-CD3depletes CCR7+DC from three aspects below:1) Since IFNγ could up-regulate Fas expression, we hypothesized that release of IFNy triggered by injection of FB anti-CD3up-regulates expression of Fas by DC, thereby inducing apoptosis and depletion of CCR7+DC. 2) Since TNFa mediates apoptosis in variety of cells and mediates cytokine storm syndrome after injection of FB anti-CD3. To confirm whether TNFa is reqiured for depletion of CCR7+DC, we injected FB anti-CD3in wild type (WT) and TNFα-/mice and checked the percentage and yields of CCR7+DC.3) Since the effectiveness on CCR7+DC depletion is different between FB anti-CD3and FNB anti-CD3IgG3, we used WT and FcR-/-mice preconditioned with FB anti-CD3to evaluate the role of FcR binding ability in depleting CCR7+DC.Our previous results have showed that depletion of CCR7+DC was related to prevention of GVHD. To further clarify the role of host CCR7+DC in the pathogenesis of aGVHD, we used CCR7-/-mice as recipients. GVHD prevention effects were compared in CCR7-/-recipients treated with or without FB anti-CD3. The classic aGVHD model in our study is C57BL/6donor'BALB/c recipient. For CCR7-/-mouse, only C57BL/6background mouse is available currently, but a stable aGVHD model using C57BL/6mice as recipients is not available. So it is crucial to build a new and stable aGVHD model with C57BL/6recipients. Megan Sykes ea t1have reported that C57BL/6recipients were transplanted with T cell depleted (TCD) spleen cells and bone marrow form B10.A donors and observed GVL effects without GVHD signs. We also used B10.A mice as donors and infused different doses of spleen cells and bone marrow to assess survival and GVHD severity in C57BL/6recipinets. Based on the data, we eventually built a stable B10.A donor'C57BL/6recipient aGVHD model. Since the effect of FcR binding ability on CCR7+DC depletion is uncertain, if CCR7+DC can not be depleted in FB anti-CD3treated FcR-/C57BL/6mice, we would inject FcR-/-C57BL/6mice with or without FB anti-CD3and do TBI and transplantation on day7after FB anti-CD3preconditioning. We would also observe suvival and GVHD severity of FcR-/-C57BL/6recipients to confirm the role of CCR7+DC in aGVHD. Although FB anti-CD3can prevent GVHD and preserve GVL effects, the side effects "cytokine storm syndrome" limit its application in clinic. To minimize the risk of cytokine storm syndrome, the dose of the FB-anti-CD3OKT3for humans approved by American FDA is limited to5mg in a75kg adult, equivalent to approximately0.1μg/g. In our murine models, the dose of FB anti-CD3is5μg/g. The dose is much higher than the approved dose0.1μg/g, making this conditioning regimen difficult to be applied in clinic. Since FNB anti-CD3IgG3causes littile "cytokine storm syndrome" and has been proved to be effective in treating autoimmune diseases, we designed sequential injection of FNB anti-CD3IgG3and FB anti-CD3. BALB/c mice were treated with FNB anti-CD3IgG3(5μg/g), and12hours later, the mice were treated with FB anti-CD3(5μg/g).7days after pre-conditioning, the mice were given TBI and transplantation. Sequential anti-CD3treatment markedly reduced the "cytokine storm syndrome", and the degree of CCR7+DC depletion and GVHD prevention is similar to the treatment of FB anti-CD3alone. All BALB/c recipients survived within100days after HSCT and did not show any signs of GVHD. To further preclinical developmet of the anti-CD3preconditioning regimen, we tried to reduce the dose of FB anti-CD3in sequeintial anti-CD3injection, or use mutilple injection of the FDA approved low dose FB anti-CD3(0.1μg/g). Thus, we wished to determine whether these preconditioning regimens could prevent GVHD in mice.Methods and materials1. WT C57BL/6mice were introvenously injected with FB anti-CD3or PBS as control.12hours later, MLN and PLN were harvested and flash frozen. Tissue sections were stained with TUNEL, goat anti-mouse CCR7and other antibodies and imaged with the use of confocal microscope. Images were taken and analyzed for expression of CCR7and apoptosis; Flow cytometry were used for detecting apoptosis of CCR7+DC, CCR7-DC as well as Fas expression.2. TNFa-/-A and FcR-/-C57BL/6mice were preconditioned with FB anti-CD3or PBS as control.7days after injection of antibody, MLN and PLN were harvested. Percentage and yields of CCR7+DC were analyzed by flow cytometry.3. WT C57BL/6recipients were conditioned with11Gy TBI.8hours later, the mice were transplanted with different doses of B10.A whole spleen cells and bone marrow to build a stable aGVHD mouse model, observe the survival and GVHD severity of recipients.4. With the B10.A'C57BL/6aGVHD model above, WT recipients were pretreated with FB anti-CD3before HSCT, and assessed for survival, GVHD severity. On day5after HSCT, expression levels of gut and skin homing and chemokine receptor by donor T cells in MLN and PLN were analyzed by flow cytometry.5. FcγRII-/-CCR7-/-C57BL/6were pretreated with or without FB anti-CD3before HSCT and assessed for survival, GVHD severity.6. With the C57BL/6'BALB/c classic aGVHD model, BALB/c mice were preconditioned with FNB anti-CD3IgG3(5μg/g) in combination with titrated dose of FB-anti-CD3of5,2.5,1.25,0.625, and0μg/g, or multiple injection of the FDA approved dose of FB-anti-CD3(0.1μg/gx5). Seven days after preconditioning, the mice were conditioned with TBI (8Gy) and transplanted. Survival and GVHD severity were assessed in C57BL/6recipients.Statistical analysisSPSS13.0software was used for statistical analysis. Measurement data was analyzed by independent-samples T test. Results were discribled as mean±standard deviation (x±s), p<0.05was considered as significant difference. Survival was analyzed by Kaplan-Meier method, and group differences were evaluated according to the log-rank test. Figures were showed with the use of Prism sofware. ResuIts1.Histology showed apoptotic CD11c+CCR7+DC clumps in MLN of WT C57BL/6mice12hours after FB anti-CD3treatment.The percentage of CD11c+DC and CD11c+CCR7+DC increased.There were no differences in the percentage of apoptotic DC substes between FB anti-CD3group and PB S group,but the yields of apoptotic CD11c+CCR7+DC significantly increased and Fas expression was up-regulated in FB anti-CD3group.2.Compared to PBS control,the percentage and yields of CD11c+CCR7+DC significantly reduced in MLN and PLN of TNFα-/-、FcγRⅡ-/-、FcγRⅡ-/-C57BL/6mice7days after FB anti-CD3preconditioning.3.It can be used as an aGVHD model when C57BL/6mice were transplanted20×106or5×106whole spleen cells and5×106bone marrow from B10.A donors.4.In B10.A'C57BL/6aGVHD model,FB anti-CD3preconditioning could prevent early aGVHD but could not prevent late GVHD after HSCT.On day5after HSCT,expression of gut homing and chemokine receptor a4β7and CCR9by donor CD4+or CD8+T cells were markedly down-regulated in MLN of FB anti-CD3preconditioned recipients,while donor T cells did not express skin homing and chemokine receptor CCR4and CCR10in PLN of both FB anti-CD3and PBS preconditioned recipients.5.GVHD was more severe in FcγRⅡ-/-and CCR7-/-C57BL/6mice as compared to WT mice.FB anti-CD3preconditioning can not prevent GVHD in FcγRⅡ-/-and CCR7-/-C57BL/6recipients.6.In the anti-CD3preconditioning regimen with sequential FNB anti-CD3IgG35μg/g and different doses of FB anti-CD3,high dose of FB anti-CD35μg/g was reqiured for preventing GVHD.Preconditioning regimen with multiple injection of low dose FB anti-CD3(0.1μg/g×5)did not have significant GVHD preventing effect in mice.Conclusion1. The mechanism of FB anti-CD3mediated CD11c+CCR7+DC depletion can be summarized as below:on the one hand, FB anti-CD3treatment triggers the release of cytokine and up-regulates expression of CCR7by DC, mediating DC migration from tissue to draining LN. Meanwhile, DC up-regulats expression of Fas, thereby increasing the sensitibity to induction of apoptosis. TNFa and other cytokines do not have direct effect on CCR7+DC depletion. On the other hand, the binding of FB anti-CD3to CD3on T cells and to FcYRIIp on CD11c+CCR7+DC activates T cells and conjoins the two cell types, inhibits the cellular activate pathway in DC, thereby inducing apoptosis of CD11c+CCR7+DC.2. A new stable B10.A'C57BL/6aGVHD model has been built. In this model, FB anti-CD3can prevent early aGVHD, but can not prevent late GVHD after HSCT.3. FB anti-CD3preconditioning can not ameliorate GVHD in FcγRⅡ-/-and CCR7-/-C57BL/6recipients. CD11c+CCR7+DC is necessary in the pathogenesis of GVHD, but is not sufficient to mediate GVHD.4. The GVHD preventing effect is dependent on the dose of FB anti-CD3.5μg/g FB anti-CD3is required for best GVHD preventing effect. Reducing the dose of FB anti-CD3is unlikely to get better results. Multiple injection of low dose FB anti-CD3has littile "cytokine storm syndrome" and shows better GVHD preventing effect as compared to one single injection of total dose of FB anti-CD3, but this preconditioning regimen is still not able to prevent GVHD effectively. Thus, we have to design a better and clinic applicable anti-CD3conditioning regimen. |
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