[The Role of NK Cells in Allogeneic Hematopoietic Stem Cell Micro-Transplantation for Acute Myeloid leukemia].

OBJECTIVE: To explore the role of NK cells in allogeneic hematopoietic stem cell micro-transplantation(MST) in the treatment of patients with acute myeloid leukemia(AML). METHODS: Data from 93 AML patients treated with MST at our center from 2013-2018 were retrospectively analyzed. The induction regimen was anthracycline and cytarabine combined with peripheral blood stem cells transplantation mobilization by granulocyte colony stimulating factor (GPBSC), followed by 2-4 courses of intensive treatment with medium to high doses of cytarabine combined with GPBSC after achieving complete remission (CR). The therapeutic effects of one and two courses of MST induction therapy on 42 patients who did not reach CR before transplantation were evaluated. Cox proportional hazards regression analysis was used to analyze the impact of donor NK cell dose and KIR genotype, including KIR ligand mismatch, 2DS1, haplotype, and HLA-Cw ligands on survival prognosis of patients. RESULTS: Forty-two patients received MST induction therapy, and the CR rate was 57.1% after 1 course and 73.7% after 2 courses. Multivariate analysis showed that, medium and high doses of NK cells was significantly associated with improved disease-free survival (DFS) of patients (HR=0.27, P =0.005; HR=0.21, P =0.001), and high doses of NK cells was significantly associated with improved overall survival (OS) of patients (HR=0.15, P =0.000). Donor 2DS1 positive significantly increases OS of patients (HR=0.25, P =0.011). For high-risk patients under 60 years old, patients of the donor-recipient KIR ligand mismatch group had longer DFS compared to the nonmismatch group (P =0.036); donor 2DS1 positive significantly prolonged OS of patients (P =0.009). CONCLUSION: NK cell dose, KIR ligand mismatch and 2DS1 influence the therapeutic effect of MST, improve the survival of AML patients.

Hematopoietic stem cell microtransplantation in patients aged over 70 with acute myeloid leukemia: a multicenter study.

In the era of molecular targeted drugs, elderly patients with acute myeloid leukemia (AML) are still very difficult to treat, especially those older than 70 years. The decline in immune function leads to serious infection and disease recurrence. The microtransplant treatment regimen (MST) chemotherapy combined with allogeneic hematopoietic stem cell infusion is a new cell therapy regimen. The aim of this MST study was to improve the survival of elderly patients by graft versus leukemia action and improving T-cell immune function. From May 2012 to July 2020, one hundred and eleven patients aged 70 to 88 years with de novo AML were analyzed retrospectively. After induction chemotherapy, patients whom complete remission (CR) was achieved were given another 2 cycles of postremission therapy. The MST groups were given allogeneic stem cell infusion after each chemotherapy cycle. CR, leukemia-free survival, and overall survival (OS) were compared between groups. Additionally, the immune function and the T cell receptor (TCR) library of T cells were detected and analyzed. The MST group exhibited an encouragingly high CR rate (63.8%), even in high-risk patients (54%), and this rate was significantly higher than that in the chemotherapy alone group. The 1-year OS of MST patients was 57.7%, and it was 55.9% in the high-risk group. It was only 37.3% in the chemotherapy alone group. Higher numbers of naive T cells were found in the MST population than in the chemotherapy alone group. More updated T-cell clones were observed in MST patients by T-cell receptor repertoire analysis with a next-generation sequencing methodology. These results suggest that MST is a safe and practical regimen conducive to longer-term survival in patients of a highly advanced age with AML. Furthermore, it has broad clinical value in the recovery of immune function in elderly patients.

Expert consensus on microtransplant for acute myeloid leukemia in elderly patients -report from the international microtransplant interest group.

Recent studies have shown that microtransplant (MST) could improve outcome of patients with elderly acute myeloid leukemia (EAML). To further standardize the MST therapy and improve outcomes in EAML patients, based on analysis of the literature on MST, especially MST with EAML from January 1st, 2011 to November 30th, 2022, the International Microtransplant Interest Group provides recommendations and considerations for MST in the treatment of EAML. Four major issues related to MST for treating EAML were addressed: therapeutic principle of MST (1), candidates for MST (2), induction chemotherapy regimens (3), and post-remission therapy based on MST (4). Others included donor screening, infusion of donor cells, laboratory examinations, and complications of treatment.

Hyper-CVAD-Based Stem Cell Microtransplant as Post-Remission Therapy in Acute Lymphoblastic Leukemia.

Post-remission strategies for patients with acute lymphoblastic leukemia (ALL) are limited to the multiagent chemotherapy and allogeneic stem cell transplant (allo-SCT), and cellular therapies are seldom involved. Although chemotherapy combined with mismatched granulocyte colony-stimulating factor mobilized peripheral blood mononuclear cell infusion (microtransplant, MST) has been studied in patients with acute myeloid leukemia, its efficacy in ALL is still undetermined. We enrolled 48 patients receiving hyper-CVAD-based MST between July 1, 2009, and January 31, 2018. No acute or chronic graft-versus-host disease occurred in patients receiving MST. Four-year overall survival (OS) and leukemia-free survival (LFS) were 62% and 35%, respectively, and the 4-year relapse rate was 65%. No patient experienced non-relapse mortality. Subgroup analysis showed that OS rates were comparable between groups with different age, risk stratification, minimal residual disease status prior to MST and immunophenotype. Adult patients tended to achieve better 4-year LFS (62% vs. 26%, P = .058) and lower hematologic relapse rate (38% vs. 74%, P = .058) compared with adolescent and young adult patients. Donor chimerism/microchimerism was detectable ranging from 0.002% to 42.78% in 78% (42/54) available samples within 14 days after each infusion and at 3 months or one year after the last cell infusion. Multivariate analyses demonstrated that white blood cells <30 × 109/L at diagnosis and sufficient hyper-CVAD cycles were prognostic factors for better 4-year OS and LFS, while the B-cell phenotype and higher number of infused CD34+ cells in the first cycle were predictors for favorable 4-year LFS. The hyper-CVAD-based MST was a feasible strategy for treating ALL patients with mild toxicity.

Rapid subcutaneous progression after immunotherapy in pretreated patients with metastatic carcinoma: two case reports.

There is heterogeneity in cancer patients' responses to immune checkpoint inhibitors (ICIs), including hyperprogression, which is very rapid tumor progression following immunotherapy, and pseudoprogression, which is an initial increase followed by a decrease in tumor burden or in the number of tumor lesions. This heterogeneity complicates clinical decisions because either premature withdrawal of the treatment or prolonged ineffective treatment harms patients. We presented two patients treated with ICIs with heterogeneous responses. One patient had Merkel cell carcinoma in the right thigh, and the other had nasopharyngeal squamous carcinoma. The first patient was treated with sintilimab and the second with sintilimab combined with abraxane. In the first patient, subcutaneous lesions grew substantially after the first cycle of treatment with sintilimab. In the second patient, subcutaneous lesions grew gradually after the second cycle of treatment with sintilimab combined with abraxane. In both cases, biopsy examination confirmed that newly emerged lesions were metastases of the primary tumor. These two cases remind clinicians that when subcutaneous nodules appear after treatment with ICIs, pathological biopsy is needed to determine the nature-pseudoprogression or rapid progression-of the disease course.

Comparison of microtransplantation, chemotherapy and allogeneic transplantation in post-remission therapy for Philadelphia chromosome-positive acute lymphoblastic leukemia.

Patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) have poor prognosis, and the efficacy of chemotherapy plus tyrosine kinase inhibitors (TKIs) followed by mismatched donor stem cell infusion (microtransplantation, MST) has not been determined. We retrospectively summarized 45 patients including 11 undergoing MST with TKIs, 17 receiving allogeneic transplant and 17 undergoing chemotherapy with TKIs. Improved 4-year overall survival rate was observed in the MST group (91%) compared with either transplant group (31%, P = .005) or chemotherapy group (36%, P = .013). The MST group also had higher 2-year and 4-year leukemia-free survival rates (91% and 72%, respectively) compared with either transplant group (33%, P = .005 and 33%, P = .021, respectively) or chemotherapy group (41%, P = .017 and 31%, P = .023, respectively). 2-year and 4-year cumulative incidences of hematologic relapse were lower in the MST group (9% and 28%, respectively) compared with those in the chemotherapy group (56%, P = .025 and 67%, P = .034, respectively). In patients undergoing MST, donor microchimerism was detected (1.07 × 10-5 to 6.6 × 10-4 copies from 9 to 1499 days) in 7 patients, and donor/patient-derived HLA*0201/2402+WT1+CD8+ T cells were found from 0.05% to 0.67% in 6 patients. MST may provide a favorable treatment for patients with Ph+ ALL.

Co-infusion of high-dose haploidentical donor cells and CD19-targeted CART cells achieves complete remission, successful donor engraftment and significant CART amplification in advanced ALL.

Autologous CD19-targeted chimeric antigen receptor-modified T cells (CD19-CART) remarkably improved the outcome of patients with advanced B-cell acute lymphoblastic leukemia (B-ALL). However, the application and outcomes of allogeneic CART cells is still uncertain. Two patients with advanced B-ALL were enrolled to receive a co-infusion of high-dose human leukocyte antigen-haploidentical donor granulocyte colony-stimulating factor mobilized peripheral blood mononuclear cells (GPBMCs; 21.01-25.34 × 108/kg) and the same donor-derived CD19-targeted CART cells (8.44-22.19 × 106/kg) without additional in vitro gene-editing following a reinduction chemotherapy as precondition. They achieved complete remission and full donor chimerism (FDC) with ongoing 20- and 4-month leukemia-free survival. A significant amplification of donor CART cells was detected in peripheral blood and/or cerebrospinal fluid and was associated with the formation of FDC. The highest amount of copies of the donor CART cells reached 4962 per µg of genomic DNA (gDNA) and 2449 per µg of gDNA, and the longest persistence was 20 months associated with B cell aplasia. Two patients experienced Grade II or III cytokine release syndromes and developed controllable Grade II intestinal acute graft-versus-host disease (GVHD) or limited chronic oral GVHD. High-dose donor GPBMC infusion may enhance amplification and persistence of haploidentical CD19-targeted CART cells, suggesting an alternative therapy for advanced B-ALL patients.

Chronic restraint stress increases social interaction in C57BL/6J mice monitoring through MiceProfiler analysis.

The social deficit is a prevailing symptom in stress-induced depression. Although social interaction behavior has been widely studied in humans and rodents, it is imprecise to record the social behavior between two free-moving mice via perusal. In the present study, we applied an approach to analyze the social behavior in mice using a software named "MiceProfiler." C57BL/6J mice were stressed via chronic restraint stress (CRS) and housed in three populations of different sizes as follows: single, three in a cage, and six in a cage. The MiceProfiler was used to analyze the video of behavioral repertoire and, the result showed that stressed and single housed mice exhibited more social interaction both in the contact time and contact activities. Furthermore, we investigated the effect of CRS on social behavior when the mice were housed in larger populations size (three or six in a cage) and found that, the CRS procedure promoted social interaction. However, the larger population size resulted in the less total contact time, less time of head-tail, and moving in an opposite way. Besides, the CRS mice showed less social avoidance while the mice from a larger population presented less active contact. And the CRS mice also exhibited a higher social hierarchy compared with the control. Our data indicated that mild restraint stress might increase the intercommunication between mice. Collectively, our findings provided a new evidence for social behavior study and the MiceProfiler could be a new tool to measure the social behaviors of rodents.

Knockdown of circHomer1 ameliorates METH-induced neuronal injury through inhibiting Bbc3 expression.

Current studies have illustrated that circular RNAs (circRNAs) are a vital part of non-coding RNA (ncRNAs) species and highly abundant and dynamically expressed in brain. However, the exact mechanisms by which circRNAs modulate methamphetamine (METH)-induced neuronal damage still remain largely unexplored. Consistent with our previous study, the expression of circHomer1 was significantly up-regulated after METH treatment in HT-22 cells. We confirmed its loop structure by detection of its back-splice junction with qRT-PCR product via sequence. Moreover, circHomer1 was resistant against RNase R digestion compared with its linear mRNA Homer1. Inhibition of circHomer1 expression indeed alleviated METH-induced neurotoxicity, with lower apoptosis rate via flow cytometry and cleaved Caspase3 protein level. Furthermore, we speculated that Bbc3 functioned as a target of circHomer1 based on computational algorithm, and knockdown of circHomer1 actually reduced Bbc3 expression at the mRNA and protein level. Besides, suppression of Bbc3 decreased the reactive oxygen species (ROS) level and radio of PI-positive cells. Furthermore, we analyzed the correlation in pairs among circHomer1, Bbc3 and behaviors in well-developed METH-addicted models using Pearson's correlation coefficient, which implied an important role of circHomer1 and Bbc3 in addictive behaviors. In all, we for the first time identified a novel circRNA, circHomer1 and our results suggested that circHomer1 regulated METH-induced lethal process by suppressing the Bbc3 expression.

Donor Selection in HLA-Mismatched Hematopoietic Stem Cell Microtransplantation for Acute Myeloid Leukemia.

To remedy the lack of human leukocyte antigen (HLA)-matched donors and address the problems bedeviling traditional allogeneic hematopoietic stem cell transplantation which induces the resultant graft-versus-host disease, we designed a scheme called HLA-mismatched hematopoietic stem cell microtransplantation (MST) for patients with acute myeloid leukemia (AML), where encouraging results were achieved. In providing answers to such questions as how to select the donors of MST and which factors were involved in the outcome of MST. One hundred thirty-one AML patients from four centers with lower or standard risk of prognosis after complete remission were given three courses of MST: high dose of cytarabine plus infusion of granulocyte colony-stimulating factor mobilized peripheral blood stem cells from HLA-mismatched donors. Leukemia-free survival (LFS) and overall survival were compared, with respect to gender difference, number of HLA-matched loci, killer cell immunoglobulin-like receptor (KIR), and ligand mismatch between donors and recipients. Median LFS of recipients with different KIR ligands from those of donors was found to be significantly higher than that of recipients having identical ligands with donors (P < 0.05). The mean LFS was statistically different between recipients whose donors had HLA-C1/C2 ligand and those whose donors had C1/C1 or C2/C2 ligand (P < 0.05). The following factors were found to promote long-term survival: female recipients of male donors' stem cell, and donors with different KIR ligands from recipients.

Comparative study of micro-transplantation from HLA fully mismatched unrelated and partly matched related donors in acute myeloid leukemia.

Micro-transplantation (MST) by chemotherapy, combined with granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (GPBSC) infusion, from an HLA partial matched related donor has shown some encouraging effective therapy for acute myeloid leukemia (AML). However, the outcome of human leukocyte antigen (HLA) fully mismatched unrelated donor-derived MST in such patients is still unknown. In the present study, we compared the efficacy of HLA fully mismatched unrelated donor-derived MST, and partly matched related donor-derived MST, in AML of 126 patients from two centers in China, These patients, aged 16 to 65 years, were given three or four courses of MST, which consisted of a high dosage cytarabine followed by GPBSC from unrelated donor or related donor. There was a statistically significant difference in 3-year leukemia-free survival (LFS) and 3-year overall survival (OS) between the unrelated and the related group. The non-treatment-related mortality (NRM) rates of patients, and other adverse complications, were no different in the two groups. In conclusion, unrelated donor-derived MST is believed to be a safe treatment, with efficacy similar to or higher than related donor-derived MST. This result provides support for the potential of MST for expanding the donor selection. However, the specific mechanism of action needs further study.

HLA-Mismatched Microtransplant in Older Patients Newly Diagnosed With Acute Myeloid Leukemia: Results From the Microtransplantation Interest Group.

IMPORTANCE: The outcome of older patients with acute myeloid leukemia (AML) remains unsatisfactory. Recent studies have shown that HLA-mismatched microtransplant could improve outcomes in such patients. OBJECTIVE: To evaluate outcomes in different age groups among older patients with newly diagnosed AML who receive HLA-mismatched microtransplant. DESIGN, SETTING, AND PARTICIPANTS: This multicenter clinical study included 185 patients with de novo AML at 12 centers in China, the United States, and Spain in the Microtransplantation Interest Group. Patients were divided into the following 4 age groups: 60 to 64 years, 65 to 69 years, 70 to 74 years, and 75 to 85 years. The study period was May 1, 2006, to July 31, 2015. EXPOSURES: Induction chemotherapy and postremission therapy with cytarabine hydrochloride with or without anthracycline, followed by highly HLA-mismatched related or fully mismatched unrelated donor cell infusion. No graft-vs-host disease prophylaxis was used. MAIN OUTCOMES AND MEASURES: The primary end point of the study was to evaluate the complete remission rates, leukemia-free survival, and overall survival in different age groups. Additional end points of the study included hematopoietic recovery, graft-vs-host disease, relapse rate, nonrelapse mortality, and other treatment-related toxicities. RESULTS: Among 185 patients, the median age was 67 years (range, 60-85 years), and 75 (40.5%) were female. The denominators in adjusted percentages in overall survival, leukemia-free survival, relapse, and nonrelapse mortality are not the sample proportions of observations. The overall complete remission rate was not significantly different among the 4 age groups (75.4% [52 of 69], 70.2% [33 of 47], 79.1% [34 of 43], and 73.1% [19 of 26). The 1-year overall survival rates were 87.7%, 85.8%, and 77.8% in the first 3 age groups, which were much higher than the rate in the fourth age group (51.7%) (P = .004, P = .008, and P = .04, respectively). The 2-year overall survival rates were 63.7% and 66.8% in the first 2 age groups, which were higher than the rates in the last 2 age groups (34.2% and 14.8%) (P = .02, P = .03, P < .001, and P < .001, respectively). The 1-year cumulative incidences of nonrelapse mortality were 10.2%, 0%, 3.4%, and 26.0% in the 4 age groups and 8.1% in all patients. The median times to neutrophil and platelet recovery were 12 days and 14 days after induction chemotherapy, respectively. Five patients had full or mixed donor engraftment, and 30.8% (8 of 26) of patients demonstrated donor microchimerism. Two patients (1.1%) developed severe acute graft-vs-host disease. CONCLUSIONS AND RELEVANCE: Microtransplant achieved a high complete remission rate in AML patients aged 60 to 85 years and higher 1-year overall survival in those aged 60 to 74 years.

[Biological characteristics of Microvesicles Derived from Bone Marrow Mesenchymal Stem Cells and Their Capacities Supporting ex vivo Expansion of Hematopoietic Stem Cells].

OBJECTIVE: To explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC). METHODS: The MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV. RESULTS: MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×106 MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34+ cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day. CONCLUSION: The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.

[Biological Characteristics of Microvesicles Secreted by Human Peripheral Blood Hematopoietic Stem Cells].

OBJECTIVE: To investigate the effects of microvesicles(MV) isolated from human peripheral blood hematopoietic stem cells(PB-HSC) on immune regulation and hematopoiesis. METHODS: PB-HSCs were separated by density-gradient centrifugation and cultrued. The supernatants of PB-HSC at 48 h were harvested for isolation and purification of MV by using ultracentrifugation. The electron microscopy was used to observe the morphology of MV. The protein level in MV was quantified through bicinchoninic acid(BCA) protein assay. Flow cytometry was used to detect the immunophenotype of MV. Human peripheral blood mononuclear cells(PB-MNC) were isolated from healthy donor and treated with isolated MV. After being co-cultured for 12 h, confocal microscopy was used to observe the action mode of MV on PB-MNC. After being co-cultured for 48 h, the levels of IL-2, IL-6, IL-8, IL-10, IFN-γ and TNF-α were detected by ELISA. Flow cytometry was used to detect the changes of T cell subsets and the activation of T cell subsets as well as intracellular cytokine staining after co-culture for 48 h. The methylcellulose was used to assess the hematopoiesis-supportive function of MV as well as co-cultured supernatants. RESULTS: The eletron microscopy revealed that MV were elliptical membrane vesicles. The protein amount in MV ranges from 29 to 110 µg. Flow cytometry showed that MV expressed mix markers on the surface, especially highly expressed MV specific marker CD63(85.86%) and hematopoietic stem cell marker CD34(33.52%). After being co-cultured for 12 h, confocal microscopy showed that MV were merged with PB-MNC. After being co-cultured for 48 h, ELISA showed that the secretion of cytokines IL-6,IL-8, IL-10 as well as TNF-α was increased while the level of IL-2 and IFN-γ was not changed much. The results of flow cytometry showed that there was no significant change in T cell subsets and T cell activation. Staining of intracellular factor showed that IL-8 was increased significantly in CD11c+ cells. The colony-forming experiments revealed that MV and the co-cultured supernatants could facilitate the colony formation. CONCLUSION: MV isolated from PB-HSC have immune-regulatery function and can prornote hematopoiesis.

[Significance of Monitoring Minimal Residual Disease by Flow Cytometry in Acute Leukemia Patients Underwent Nonmyeloablative Allo-HSCT].

OBJECTIVE: To explore the value of dynamically monitoring minimal residual disease (MRD) by flow cytometry before and after non-myeloablative allo-HSCT (NST) for prediction of acute leukemia(AL) relapse after transplantation. METHODS: The clinical data of 51 AL patients underwent NST were analyzed retrospectively in Department of Hematology of Affiliated Hospital of Academy of Military Medical Sciences from January 2011 to December 2015. All AL patients achieved the morphologic complete remission of bone marrow before transplantation. The bone marrow samples were collected for monitoring of MRD within 35 days before transplant, every month till 3 months after transplant, every 3 months till 24 months after transplant, and then every 6 months after 2 years of transplant. According to the MRD cutoff value of 0.2%, the AL patients were divided into high level MRD group (18 cases) which was defined as MRD≥0.2% after transplantantion at least for 1 time, and low level MRD group (33 cases) which was defined as MRD<0.2% after transplant all the time. 2 year cumulative relapse rate in 2 groups were compared. RESULTS: Two-year relapse rates were 6.1% and 50% in low-level MRD group and high-level MRD group post NST(P=0.001)respectively. Multivariate analysis indicated that the risk of relapse in high level MRD group was 5.84 times of low level MRD group(P=0.036). MRD≥0.2% post transplant was an independent risk factor for leukemia relapse post NST. The mortality rate was 81.8% and 46.3%(P<0.05) in relapse and non-relapse groups respectively. CONCLUSION: Dynamically monitoring MRD by FCM is a crucial tool for early relapse estimation of acute leukemia in adult patients after allogeneic nonmyeloablative hematopoietic stem cell transplantation. MRD≥0.2% after transplant can be used as a early valuable evidence for predicting relapse and guiding active medical intervention.

[Effect of Infusion of Recipient Spleen Cells at Different Time after Murine Haploidentical Hematopoietic Stem Cell Transplantation on Graft Versus Host Disease].

OBJECTIVE: To explore the effect of infusing G-CSF mobilized recipient spleen cells at different time after haploidentical stem cell transplantation(HSCT) on graft-versus-host disease (GVHD) in mice and its possible mechanism. METHODS: Forty mice after HSCT were randomly divided into 4 groups (n=10): GVHD positive control group (control group), 1st d recipient cell infusion group after transplantation (+1 d group), 4th d recipient cell infusion group after transplantation(+4 d group), 7th d recipient cell infusion group after transplantation(+7 d group). The mice in control group were injected the normal saline of same equivalent with experimental group which were given the same amount of G-CSF-mobilized recipient spleen cells. The general manifestation and pathological change of GVHD were observed. The expression changes of CD3+CD4+, CD3+CD8+ cell subsets and FasL in peripheral blood were detected by flow cytometry. RESULTS: The incidence of GVHD was significantly decreased in +4 d group and the median survival time was longer than 60 days, which was significantly higher than that of control group (24 d), +1 d group (21 d), +7 d group (28 d). (P<0.01, P<0.01, P<0.01). The Fasl expression of peripheral blood T lymphocytes in +4 d group were significantly lower than that in the other 3 groups(P<0.05). CONCLUSION: The +4 d infusion of G-CSF mobilized recipient spleen cells on 4th day after haploidentical HSC transplantation can inhibit the expression of FasL in donor T lymphocytes, and significantly reduce the incidence of GVHD.

[Establishment of Mouse Model of H-2 Haploidentical Hematopoietic Stem Cell Transplantation from Double Donors].

OBJECTIVE: To establish a new mouse model of H-2 haploidentical stem cell transplantation from double donors (DHSCT) and compare with conventional haploidentical hematopoietic stem cell transplantation (HSCT) so as to alleviate transplant-related complications. METHODS: The recipients CB6F1 of conventional HSCT group were pretreated by 8 Gy total body irradiation(TBI), and received 3×107 donor (male C57) spleen mononuclear cells (spMNC) mobilized by G-CSF within 2 hours after TBI. Recipients CB6F1 of D-HSCT groups accepted 2 Gy TBI, and received total 12×107 spMNC mobilized by G-CSF from 2 donors within 2 hours after TBI, each donor donated 6×107 cells. According to the different strains and sex of donors, DHSCT were divided into 3 groups: in group A, the stem cells were from male C57 and female BALB/c; in group B, stem cells were from male C57 and male BALB/c, while the stem cells in group C were from male C57 and male C3H. Hematopoietic reconstruction, engraftment, GVHD and survival were observed among these 4 groups. RESULTS: The nadir of white blood cell count after conventional HSCT were lower than 1×109/L and lasted for 3 to 5 days, while not less than 3×109/L after D-HSCT among either group A, B or C. The complete chimerism (CC) in conventional HSCT group was achieved quickly within only 1 week in peripheral blood. Mixed chimerism (MC) in peripheral blood was found within the first week after DHSCT among either group A, B or C, and transformed into stable CC within the second week eventually. Both GVHD morbidity and mortality of conventional HSCT were 100% at 34th day after transplantation.Among DHSCT groups,the overall GVHD morbidity and mortality at 34th day after transplant were 49.6% and 50%(P<0.01,P<0.05), respectively,and 60.4% and 81.2% at 50th day after transplant. Overall survival of 50 days was 50.9% that indicated a long survival in such mice DHSCT. The differences of hematopoietic reconstruction, donor cell engraftment, GVHD incidence, GVHD mortality and OS were not statistically significant among group A, B and C(P>0.05). CONCLUSION: A new mouse model of H-2 haploidentical peripheral blood stem cell transplantation from double donors (DHSCT) has been successfully established by reducing conditioning intensity and increasing graft cell numbers from double haploidentical donors without GVHD prophylaxis. DHSCT successfully achieved stable complete chimerism, less GVHD morbidity and mortality and longer OS without hematopoietic suppression. This study provides experimental evidence for clinical application of HLA haploidentical peripheral blood stem cell transplantation from double donors.

The long-term outcome of reduced-intensity allogeneic stem cell transplantation from a matched related or unrelated donor, or haploidentical family donor in patients with leukemia: a retrospective analysis of data from the China RIC Cooperative Group.

This study compared 6-year follow-up data from patients undergoing reduced-intensity conditioning (RIC) transplantation with an HLA-matched related donor (MRD), an HLA-matched unrelated donor (MUD), or an HLA-haploidentical donor (HID) for leukemia. Four hundred and twenty-seven patients from the China RIC Cooperative Group were enrolled, including 301 in the MRD, 79 in the HID, and 47 in the MUD groups. The conditioning regimen involved fludarabine combined with anti-lymphocyte globulin and cyclophosphamide. Graft-versus-host disease (GVHD) prophylaxis was administered using cyclosporin A (CsA) and mycophenolate mofetil (MMF). Four hundred and nineteen patients achieved stable donor chimerism. The incidence of stage II-IV acute GVHD in the HID group was 44.3 %, significantly higher than that in the MRD (23.6 %) and MUD (19.1 %) groups. The 1-year transplantation-related mortality (TRM) rates were 44.3, 17.6, and 21.3, respectively. Event-free survival (EFS) at 6 years in the HID group was 36.7 %, significantly lower than that of the MRD and MUD groups (59.1 and 66.0 %, P < 0.001 and P = 0.001, respectively). For advanced leukemia, the relapse rate of the HID group was 18.5 %, lower than that of the MRD group (37.5 %, P = 0.05), but the EFS at 6 years was 31.7 and 30.4 % (P > 0.05), respectively. RIC transplantation with MRD and MUD had similar outcome in leukemia which is better than that with HID. RIC transplantation with HID had lower relapsed with higher TRM and GVHD rate, particularly in advanced leukemias. RIC transplantation with MRD and MUD had similar outcomes in leukemia and they were better than those with HID. RIC transplantation with HID had a lower relapse rate but higher TRM and GVHD rates, particularly in cases of advanced leukemia.

[Features of Immunophenotypes and Characteristics of Molecular Biology and Cellular Genetics of AML Patients with CD4 and CD7 Expression].

OBJECTIVE: To explore the features of immunophenotypes and the characteristics of molecular biology and cellular genetics of AML patients with CD7 and CD4 expression. METHODS: The immunophenotypical markers of AML cells were detected by multiple parameter flow cytometry; the expression of WT1, MDK, ETO, PML-RaRa and BCR-ABL were detected by RT-PCR; and cellular features were analyzed by R-band in 304 patients. The patients were divided into three groups according to their immunophenotypes: AML with CD7 expression (CD7 group), AML with CD4 expression(CD4 group) and AML without CD7 and CD4 expression (common AML group). RESULTS: The expression rate and level of HLA-DR in CD7 group were higher than those in the common AML group, and the expression rate of CD33 and CD34 was higher than that in the other two groups. The expression rate and level of CD15, CD64 in the CD4 group were higher than those in the other 2 groups, and the expression rate and level of CD33 were higher than those in the common AML group. WT1 expression in the CD7 group was lower than that in the common AML group. PML-RaRa was not detected in the CD7 group. AML with co-expression of CD4 or CD7 showed more normal karyotype. (15;17) was not found in AML with CD7 expression. CONCLUSION: AML cells with CD7 expression originate from precursor cells and are blocked in the early phase of hematological development; AML cells with CD4 expression originate from more mature stage of hematological devevelopment and with CD33, CD64 and CD15 high expression; AML cells with CD7 and CD4 expression are characterized by no-specific change of cellular genetics. According to the expression level and intesity of CD4 and CD7, and together with other specific lineage markers, the MRD in AML patients can be quantitatively detected.

Co-infusion of haplo-identical CD19-chimeric antigen receptor T cells and stem cells achieved full donor engraftment in refractory acute lymphoblastic leukemia.

BACKGROUND: Elderly patients with relapsed and refractory acute lymphoblastic leukemia (ALL) have poor prognosis. Autologous CD19 chimeric antigen receptor-modified T (CAR-T) cells have potentials to cure patients with B cell ALL; however, safety and efficacy of allogeneic CD19 CAR-T cells are still undetermined. CASE PRESENTATION: We treated a 71-year-old female with relapsed and refractory ALL who received co-infusion of haplo-identical donor-derived CD19-directed CAR-T cells and mobilized peripheral blood stem cells (PBSC) following induction chemotherapy. Undetectable minimal residual disease by flow cytometry was achieved, and full donor cell engraftment was established. The transient release of cytokines and mild fever were detected. Significantly elevated serum lactate dehydrogenase, alanine transaminase, bilirubin and glutamic-oxalacetic transaminase were observed from days 14 to 18, all of which were reversible after immunosuppressive therapy. CONCLUSIONS: Our preliminary results suggest that co-infusion of haplo-identical donor-derived CAR-T cells and mobilized PBSCs may induce full donor engraftment in relapsed and refractory ALL including elderly patients, but complications related to donor cell infusions should still be cautioned. TRIAL REGISTRATION: Allogeneic CART-19 for Elderly Relapsed/Refractory CD19+ ALL. NCT02799550.