| Background and ObjectiveHematopoietic stem cell transplantation (HSCT) is a curative therapy of hematological malignancies and hereditary diseases. Since the first HSCT was successfully performed in 1965, HSCT has achieved a great progress in recent decades. To allow stem cells from donor engraft in bone marrow to reconstitute hematopoietic and immunity, intensive conditioning was essential to destroy the normal hematopoietic and immune function of patients. The hematopoietic reconstitution recovered fast in short time, but the time of immune reconstitution needs a long time. The delay of immune reconstitution after transplant results in inferior immune function, and patients are prone to be infected and relapse after transplant. The infections and relapse are the main death reasons for patients after transplant.Immune reconstitution included the reconstitution of the cellular immunity and humoral immunity, and the reconstitution of the cellular immunity is most important. The neutrophil recovery after Hematopoietic stem cell transplantation was first completed, the normal neutrophil recovery allows recipients to have certain anti-infection immunity. The time of neutrophil recovery was about 14-21 days. Followed by the recovery of natural killer (NK) cells and mononuclear cells, it takes 14-60 days for the two cells recovering to the normal level after transplantation, The normal count of NK cells allow recipients to have certain antiviral immune function, but the mature of normal function of NK cells takes a long time. The recovery of CD8+ T cells and B cells lasts 2-8 months, the normal number of B cells can produce antibodies in patients, but the normal of humoral immune function still needs a long time, probably 1-2 years. CD4+ T cells is the main effect cell of cytotoxic, it has a special ability to anti-bacterial infection and an immune surveillance function to erase the mutated cells. The recovery of CD4+ cell was slowest after transplant, it takes 1 to 2 years for patients undergoing full matched HSCT to recover and more than five years for the patients undergoing haploidentical HSCT.There are two ways of the Τ-cell immune reconstitution:(1) Thymus-dependent pathway:the quick growth of peripheral lymphocytes originated from the naive Τ-cell and memory Τ-cell in the peripheral blood. These two kinds of cells are from the mature Τ-cells in grafts from donor and the residence of Τ-cells in recipients after conditioning regimen. However, this pathway can’t produce new naive Τ-cells and it lasts only a short time after transplant. For a complete function of adaptive immunity, the new native Τ-cells should be produced continuously and well matured in peripheral lymphoid organs. (2) Thymus-dependent pathway:In normal body, the location of the development and mature of Τ-cells is in thymus. The hematopoietic stem cells differentiated to lymphatic progenitor cells in bone marrow, then the lymphatic progenitor cells migrated to thymus to undergo negative and positive selection in thymus. After negative and positive selection in thymus, the Τ-cell can achieve the ability to limited recognize the MHC and keep the tolerance of self-antigen. Only 5% new naive Τ-cell can be produced to migrate to peripheral blood from the thymus. The thymus function plays an important role in Τ-cell immune reconstitution.It’s a problem to know the exact immune reconstitution in patients. The development of flow cytometry technique (FCM) can help test the subgroup of Τ-cells. However, it’s hard for the technique of FCM to know the origin of Τ-cells. Τ-cell receptor excision circle (TREC) is a circle DNA produced by the TCR-a gene during the development of Τ-cells. TREC can’t amplify in cell and be diluted in cell. The count of TREC can present number of native Τ-cells with functional TCR gene. The copies of TREC can be tested and calculated by real-time PCR technique. This test can directly evaluated the cell count of native Τ-cells produced by thymus and we can know the state of the thymus and new naive Τ-cell.It’s reported that the conditioning regimen, graft versus host disease (GVHD), the active disease at transplant and the component of grafts are the main reasons that effect the central immune function after transplant. Myelablative conditioning regimen mainly influenced the quantity and function of thymic epithelial cell (TEC) and also reduced the cytokines and chemokine which were the essential stuff of proliferation, differentiation and migration of lymphatic progenitor cells. Experts have found that the radiation dose before transplant will significantly influence the quantity and quality of naive Τ-cell that produced by thymus in mice. It’s probably the change or deficiency function of thymic stroma cell induced the change of the quantity and function of TEC. At the same time, the cytokines and chemokine which were the essential stuff of proliferation, differentiation and migration of lymphatic progenitor cells reduced. GVHD can influence the morphological change of thymus. It will reduce the thymocyte and change the component of stroma cells. It has been testified that the GVHD can damage the output function of thymus by testing the TREC copies. It also can disturb the mature of thymocyte. In the mice model of GVHD, the deficiency of selection of development and differentiation of Τ-cell may associate with the change of component and structure of TEC.Although many patients were cured by undergoing the HSCT in our center basing on the myelablative conditioning regimen of FBCA which was set up in 1998. The long-term survival of patients with refractory hematological malignancies was 35% and that of patients with intermediate risk was 60% in our center. However, the relapse and infection after transplant are still the main reason of death. In our center, the rate of relapse and infection were 20% and 60%, respectively. How is the central immune reconstitution in this conditioning regimen? What’s the relationship between infection and central immune after transplant? It is unclear. The patients underwent HSCT with hematological disease were enrolled in our study. We evaluated the quantity of Τ-cell and TREC in different time after transplant and analyzed the relationship between the infection and central immune after transplant.Methods1. PatientsPatients with hematological disease undergoing HSCT in department of hematology in our hospital between 2013 and 2014. There are totally 20 patients enrolled in study. The median age was 18 (3-47),12 patients were younger than 20, and 8 patients were older than 20. There are 11 men and 9 women in our cohort. The diagnose of disease in our cohort:6 patients with acute myeloid leukemia (AML); 4 patients with acute lymphocyte leukemia (ALL); 2 patients with chronic myeloid leukemia (CML); 5 patients with severe aplastic anemia (SAA); 2 patients with thalassemia; 1 patient with myedysplastic syndrome (MDS).7 patients were categoried as intermediate risk group, while 13 patients were classified as high-risk group.2. HSCTThe health relatives of patients are the resources of donor. The HLA type was assessed by sequenceing-based typing (SBT) or sequence-specific primers PCR (PCR-SSP).20 patient underwent HSCT basing on our FBCA conditioning regimen, which consisted of fludarabine (25 mg/m2/day, intravenous) on days -9 to -5, busulfan (3.2 mg/kg/day, intravenous) on days -8 to -5, cyclophosphamide (60 mg/kg/day, intravenous) on days -3 to -2, and rabbit anti-lymphocyte globulin (ATG, Thymoglobulin, Genzyme,2.5 mg/kg/day for haploidentical and 1.25mg/kg/day for full matched, intravenous) on days -5 to -1. The G-CSF primed peripheral blood stem cell was the component of the graft. All patients were given a combination of cyclosporine A (CsA) and short-term methotrexate (MTX) for GVHD prophylaxis. CsA was administered (3 mg/kg/day) by continuous intravenous infusion on day-1 and switched to oral CsA as soon as patients were off intravenous therapy. The CsA should be tapered gradually and stopped after 180 without GVHD. All patients received MTX (15 mg/m2/day, day+1; 10 mg/m2/day, days +3,+6,+11) after transplantation. The G-CSF and TPO were used for stimulating the hematopoietic reconstituon after transplant.3. The evaluation of Τ-cell subsets.The Τ-cell subset was evaluated by FCM among 20 patients after 1 month,3 month,6 month and 12 month transplant to know the quantity of CD4+ and CD8+ in peripheral blood.4. The evaluation of TREC copiesThe peripheral mononuclear cell of 20 patients were collected afterl month,3 month,6 month and 12 month transplant. The DNA was extracted by test kits. The TREC copies in 100000 cells were calculated by real-time PCR to evaluate the function of thymus.5. The relationship between infection and immune reconstitution after transplant.To systemize and analyze the quantity of Τ-cell subset and copies of TREC in different time after transplant. The SPSS17.0 was used to analyze the data. P value<0.05 is significant.Results1. All patients achieved myeloid, platelet reconstitution and steady full-donor chimerism after transplantation. The median time for neutrophil engraftment was 13 days (range,11-18 days); for platelet recovery, it was 14 days (range, 9-20 days).6 patients had aGVHD grade I and 1 patient had grade II after 100 days transplant.3 patients achieved limited chronic GVHD (cGVHD), and 1 patient had severe liver GVHD. Only 1 patient died in this group, the death reason was relapse and GVHD grade IV of liver.2. The absolute count of CD3+ Τ-cell of all patients was 339.72±69.79/μL in 1 month after transplant, then it raised to 405.88±85.68/μL in 3 months after transplant.It continued to rise to 810.05±208.11/μL and 1207.99±250.72/μL in 6 months and 12 months after transplant, respectively. When it comes to CD4+ Τ-cell, the absolute count of CD4+ Τ-cell of all patients was 138.98±41.96/μL in 1 month after transplant, then it continued to reduce to 126.50±17.02/μL in 3 months after transplant. It started to rise to 228.91±66.95/μL and 358.14±195.46/μL in 6 months and 12 months after transplant, respectively. The absolute count of CD8+ Τ-cell were 255.63±47.23/μL,371.91±62.69/μL, 775.82±73.26/μL and 971.87±138.56/μL in 1 month,3 months,6 months and 12 months after transplant, respectively. The counts of Τ-cell sunset of patients in age≥20 year group were lower than the younger group, especially in the counts of CD4+ Τ-cell in 6 months and 12 months after transplant (P value were 0.028 and 0.0001, respectively).3. The copies of TREC of all patients were 0.93±2.45 copies/105 cells,7.56±7.64 copies/105 cells,85.83±54.80 copies/105 cells and 148.62±77.61 copies/105 cells in 1 month,3 months,6 months and 12 months after transplant. However, the copies of TREC in all patients were lower than normal people comparing to the other reports. The copies of TREC of patients in age≥20 year group were lower than the younger group, especially in t 6 months and 12 months after transplant (P value were 0.012 and 0.002, respectively).4. No matter in which transplant type, the counts of CD4+ Τ-cell in 12 months and copies of TREC in 6 months and 12 months after transplant were higher in younger age group (P value<0.05).5.17 patients got infection after transplant. In 6 months of younger group after transplant, there are 12 EB infection,13 CMV infection,3 hemorrhagic cystitis, 2 herpes virus and 1 fungal infection of lung. In 6 months of older group after transplant, there are 5 EB infection,5 CMV infection,4 hemorrhagic cystitis,1 herpes virus and 1 fungal infection of lung. After 6 months transplant, only 1 patients got fungal infection in younger group, while 5 patients in older group got that infection.Conclusion1. The transplant protocol with FBCA myelablative conditioning regimen and G-CSF primed peripheral blood stem cell without in virto Τ-cell depletion we developed is feasible and well-tolerated, the central immune reconstitution after transplant ws comparable to those of contemporaneous transplant performed by other centers.2. In different transplant type, the age is an important factor that affect the central immune reconstitution. The immune reconstitution of younger group was fast than the older. The fungal infection in the later time after transplant is less in younger group, and the immune reconstitution in patients with fungal infection was lower than the one without fungal infection. This may suggest the less fungal infection in younger group associated with well function of thymus and rapid immune reconstitution in young people.3. The recent output of thymus in patients undergoing HSCT is lower than the normal people. However, the function of thymus had a close relationship with the central immune reconstitution after transplant. To improve the function of thymus may be the way to overcome the delayed immune reconstitution after transplant. |
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