| Background and ObjectiveAllogeneic hematopoietic stem cell transplantation (allo-HSCT) has became a potentially curative therapy of a wide variety of hematological malignancies and nonmalignancies, and major indications include leukemia, lymphomas, and bone marrow failure. Since the first HSCT was successfully performed in the late 1950s, more than 1 million procedures have been completed worldwide. With the deepen understanding of the human leukocyte antigen (HLA), graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect, HSCT has achieved a great progress and the long-term survival of patients with leukemia have reached 40%-60%. However, the delay of immune reconstitution after HSCT, especially the delay of central immune reconstitution, lead to opportunistic infection and relapse of leukemia after transplantation, and affects the long term outcome of HSCT. Thus, it is urgent to improve the immune reconstitution after transplantation, reduce the time of immune deficiency, and reduce the relapse after transplantation.There are two parts of the immune reconstitution after HSCT:(1) innate immune reconstruction includes the reconstitution of natural killer cells (NK), and dendritic cells (DC) etc.; (2) adaptive immune reconstruction includes the reconstitution of T and B lymphocytes. The NK cells recovery after HSCT was first completed. The time of recovery is about 1-2 months and CD56bright CD16dim is the main subsets in the early time. Followed by the recovery of B cells, it usually takes 2-8 months for the recovery in number, and the recovery in function of producing immunoglobulin (Ig) requires 1-2 years. T cell recovering in number and function need a long time than others. The reconstruction of CD8+T cells lasts 2-8 months for recovering to the normal level after transplantation. The recovery of CD4+T cells is the slowest after transplantation, it generally need 1-2 years to fully recover, sometimes even longer than that.Thymus is a central immune organ, mainly responsible for the generation and output of naive T cells, and plays an important role in T-cell immune reconstitution after transplantation. Lymphatic progenitor cells, which derived from the differentiation of hematopoietic stem cells in bone marrow, migrated to thymus to differentiate three-negative cells (TNC) in the thymic cortex. TNC became single-positive T cells (SP) after undergo negative and positive selection in the thymus. Then T cells can achieve the ability to limited recognize the MHC and keep the tolerance of self-antigen. After the development process, only about 5% naive T cell can be produced and output to circulation and peripheral lymphoid organs. Thymic epithelial cells (TECs), DC, macrophages, thymic stromal cells and extracellular matrix constitute a specific three-dimensional structure, called thymic microenvironment, which play a key role on T cell development. TECs which include cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are the main cells in thymic microenvironment. TECs directly contact T cells and provide it stimulatory signals to promote the development of T cells in the thymus. Obviously, thymus (especiallyTECs) plays a crucial role on T cells immune reconstitution.The thymus is very sensitive to the immune injury. Both high dose radiotherapy and chemotherapy, the use of immunosuppressant after HSCT, and GVHD can get thymus injury, especially TECs, and affect the function of it. Total body irradiation (TBI) can significantly reduced the number of TECs which can produce cytokines and chemokines for the T cell development. Myeloablative regimens probably influence the function of thymic stromal cells, so that the quantity and quality of thymus lymphocytes decreased significantly after transplantation. GVHD can also decrease thymic cells and change the components of stromal cells to affect the development, differentiation and output of T cells. In addition, as the age grows, the differentiated capacity of hematopoietic stem cells weakened, lymphoid progenitor cells decreased, and thymus degradated. These significantly delayed the immune reconstitution in elderly patients after HSCT.Several strategies have been proposed for protecting and restoring thymic function, and boosting the immune reconstitution after allo-HSCT, mainly including cytokines, hormones and cell therapy. Keratinocyte growth factor (KGF) administration enhance thymopoiesis and peripheral T cell reconstitution after syngeneic or allogeneic HSCT by promoting the proliferation of TECs and protecting them from damage caused by radiation, chemotherapy or GVHD. IL-7 is a lymphopoietic cytokine that can directly promote lymphocyte development. It increases the speed of T cell reconstitution after syngeneic or allogeneic HSCT by promoting the proliferation of lymphocytes and lymphoid precursors. Administration of growth hormone (GH) in mice after HSCT improves thymic cellularity, protects bone marrow from radiation injury, delays thymic involution and positively regulates T cell migration. Insulin like growth factor 1 (IGF-1) and growth hormone secretagogue (GHSs) promote the proliferation of thymic epithelial cells and improve the immune reconstitution probably by promoting secretion of GH.Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), with a potent circulating orexigen, controlling energy expenditure, adiposity, and growth hormone secretion. Ghrelin combined to GHS-R which expressed on the surface of T cells, B cells, mononuclear cells and other immune cells to controlling cytokine expression, and cellular activation, trafficking, and apoptosis. Ghrelin not only increased the number and weight of thymocytes, but also increased the numbers of TECs, early thymocyte progenitors (ETPs), pluripotential stem cells and the common lymphoid progenitors in the thymus and bone marrow, respectively. Moreover, Ghrelin administration significantly improved the thymic architecture resulting in much more defined cortical and medullary regions and increased the number of thymic and peripheral recent thymic emigrants in aged mice. Together, these results suggest that Ghrelin and its receptor play a key role in thymic biology and T-cell development.Although the study of Ghrelin on thymus is mainly focused on the aged mice, not much on immune reconstitution after allo-HSCT, the effects on thymus and T cell of Ghrelin may also occur on immune reconstitution. Whether the GH-IGH-1 aixs play a role? What are the difference effects between the GH and Ghrelin on the immune reconstitution after allo-HSCT? These are unclear. Therefore, we establish a model of allogeneic bone marrow transplantation (allo-BMT) with mice and administered Ghrelin at different time. The thymus index, spleen index, the quantity of T cell subsets (CD4+/CD8+) and the expression of TREC were measured at different time after BMT to study the effect of Ghrelin on the T-cell immune reconstitution.Methods1. A model of allo-BMT with mice was established:donor were C57BL/6 mice (H-2Kb), recipient were BALB/c mice (H-2Kd). First, we found out the smallest myeloablative radiation dose. Then we found out the optimal number of bone marrow cells for allo-BMT.2. Experiments were carried out on the basis of allo-BMT model. Mice were divided into three groups. GG group:20?g/d Ghrelin were administered intraperitoneally on days -7 to 28. PG group:20?g/d Ghrelin were administered intraperitoneally on days 1 to 28. PP group:0.2ml/d PBS were administered intraperitoneally on days -7 to 28.3. Mice were observed everyday to evaluate its performance and clinical manifestation of GVHD, measured weight twice a week. Thymus and spleen index, histologic section of thymus, donor chimerism, T-cell subset and TREC expression were detected after 14 days,21 days and 28 days BMT.Results1. Mice with 7.0Gy TBI could live more than 60days, while mice with 7.5Gy, 8.0Gy,8.5Gy TBI were died of hematopoiesis failure.7.5Gy was the minimum lethal dose and performed as myeloablative conditioning regimen in our study. Mice infused with 2×107 bone marrow cells for transplantation have a higher rate of donor chimerism than infused with 5×106 bone marrow cells (P>0.05).2. The weight of mice in the GG, PG and PP group were all decreased after BMT, and began to increase after 7 days transplant. The change of weight in GG, PG and PP groups have no significant difference (P>0.05).3. The donor chimerism rate reached more than 90% in each group. There was no skin injury, diarrhea and other manifestations of GVHD. Histologic sections of skin, liver and small intestine have no lymphocyte infiltration.4. The thymus and spleen index were lower than the normal level after BMT. There was no significant difference between three groups in the same time period (P> 0.05). In the same group, thymus and spleen index showed no significant difference on 14 days,21 days and 28 days (P> 0.05).5. Mice in GG group significantly improved the thymic architecture resulting in much more defined cortical and medullary region, and increased the thymic cellularity on 14 days,21 days and 28 days. These effects found in PG group on 28 days after BMT. In PP group, although the thymic celluarity improved with the time, thymic architecture has not been restored resulting in undefined cortical and medullary region until 28 days.6. In GG group, the absolute count of CD4+T cells was significantly higher than that of PG and PP group (P< 0.05). And the difference was more obvious on 28 days. The counts of CD4+T cell in GG group has reached 22.03±5.40×105, almost close to normal level. Compared with the PP group, the absolute counts of CD4+T cells in PG group on 28 days after transplantation was higer and reached 13.97±3.58×105 (P< 0.05). The absolute counts of CD8+T cells were no significant difference between the three groups (P>0.05).7. On 14 days after transplantation, the expression of TREC in spleen mice were unable to detected, and the expression of TREC in thymus is lower than 21 and 28 days. On 21 days, the expression of TREC in spleen and thymus increased, and TREC of thymus in GG, PG and PP group were 0.0155±0.0101?0.0133 ±0.011?0.0105±0.0056, respectively. TREC expression have no significant difference in three different treatment groups (P>0.05). However, the expression of TREC in the thymus was significantly higher than that in the spleen (P< 0.05).Conclusion1. The minimum lethal dose of TBI is 7.5Gy. The optimal number of bone marrow cells is 2×107. A model of allo-BMT with mice was established without GVHD and is good model for the study on immune reconstitution after transplantation.2. Within 28 days after allo-BMT, injection of Ghrelin has no effect on body weight and the expression of TREC in the thymus and spleen, and it can not improve output of naive T cells. It should take a further study to figure out the effect of Ghrelin on immune reconstitution more than 28 transplant days.3. Both continuous injection of Ghrelin on days-7 to 28 or days 1 to 28, improved the thymic architecture resulting in much more defined cortical and medullary region, enhances the thymic cellularity, and increases the absolute count of splenic CD4+T cell. The study suggests that Ghrelin can promote immune reconstitution after allo-BMT in mice, and Ghrelin injection before transplantation protect thymus from TBI injury in some degree. The promoting effect of Ghrelin on immune reconstitution may become a new way to overcome the delayed immune reconstitution after HSCT. |
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