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.

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.

Proceedings from the Second Haploidentical Stem Cell Transplantation Symposium-Haplo2014, San Francisco, California, December 4, 2014.

Significant progress has been made over the past decade in haploidentical transplantation, with the development of novel methods to control intense alloreactive reactions generated in the major HLA-mismatched setting. Application of post-transplantation cyclophosphamide has gained worldwide acceptance as an effective and low-cost way to perform this type of transplantation, with outcomes now similar to those from HLA-matched unrelated donors. These advances have resulted in improved treatment-related mortality, whereas disease relapse has emerged as the most common cause of treatment failure. In addition, improvements in immunologic reconstitution after transplantation are much needed, not only in haploidentical transplantation but in all forms of stem cell transplantation. This symposium has focused on some of the most promising methods to control alloreactivity in this form of transplantation and application of cellular therapy to prevent disease relapse after transplantation, as well as understanding immunologic reconstitution and foreseeable approaches to improve immune recovery after transplantation.

[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.

HLA-mismatched allogeneic adoptive immune therapy in patients with severely immunosuppressed AIDS: a multicenter, open-label, controlled, phase 2a study.

Recurrent opportunistic infections (OIs) in patients with severely immunosuppressed AIDS remain an unresolved medical challenge despite advancements in antiretroviral therapy (ART). To address this gap, we developed an HLA-mismatched allogeneic adoptive immune therapy (AAIT) specifically targeting this patient population. The safety and efficacy of this novel therapeutic approach were preliminarily confirmed in our phase 1 trial. Subsequently, a multicenter, open-label, controlled, phase 2a trial was conducted to evaluate the efficacy of AAIT in combination with ART compared with the conventional ART-only regimen. No difference in the incidence of adverse events (AEs) was observed between the two groups at the 96-week follow-up. AAIT treatment improved CD4+ T cell recovery at weeks 72 (P = 0.048) and 96 (P = 0.024) compared to the Control Group. Additionally, stratified analysis of patients in the AAIT Group showed that donor/recipient sex mismatch was significantly associated with the likelihood of patients achieving an immunological response (OR = 8.667; 95% CI, 2.010-37.377; P = 0.004). These findings suggest that AAIT serves as a promising adjunct therapy for improving the outcomes of patients with severely immunosuppressed AIDS. Further studies are needed to elucidate the immunological mechanisms underlying AAIT and identify the subpopulations that respond optimally to this therapeutic approach. This trial is registered at www.clinicaltrials.gov (NCT04098770).Trial registration: ClinicalTrials.gov identifier: NCT04098770.Trial registration: ClinicalTrials.gov identifier: NCT02651376.

Infusion of HLA-mismatched peripheral blood stem cells improves the outcome of chemotherapy for acute myeloid leukemia in elderly patients.

Treatment outcome of acute myeloid leukemia (AML) in elderly patients remains unsatisfactory. It has been shown that the infusion of granulocyte colony-stimulating factor-mobilized donor peripheral blood stem cells (G-PBSCs) can enhance graft-versus-leukemia effects and speed hematopoietic recovery. Fifty-eight AML patients aged 60-88 years were randomly assigned to receive induction chemotherapy with cytarabine and mitoxantrone (control group; n = 28) or it plus human leukocyte antigen-mismatched G-PBSCs (G-PBSC group; n = 30). Patients who achieved complete remission received another 2 cycles of postremission therapy with intermediate-dose cytarabine or it plus G-PBSCs. The complete remission rate was significantly higher in the G-PBSC group than in the control group (80.0% vs 42.8%; P = .006). The median recovery times of neutrophils and platelets were 11 days and 14.5 days, respectively, in the G-PBSC group and 16 days and 20 days, respectively, in the control group after chemotherapy. The 2-year probability of disease-free survival was significantly higher in the G-PBSC group than in the control group (38.9% vs 10.0%; P = .01). No graft-versus-host disease was observed in any patient. Persistent donor microchimerism was successfully detected in all of the 4 female patients. These results indicate that G-PBSCs in combination with conventional chemotherapy may provide a promising treatment method for AML in elderly patients.

[Clinical investigation of refractory lymphoma with HLA-mismatched stem-cell microtransplantation].

OBJECTIVE: To explore the efficacy and safety of HLA-mismatched stem-cell microtransplantation in patients with refractory lymphoma. METHODS: This study included 10 patients with relapsing or refractory lymphoma at Department of Hematology, Second Artillery General Hospital from October 2009 to February 2012. All patients received programmed infusions of G-CSF-mobilized HLA-mismatched donor peripheral blood stem cell (G-PBSC) after each cycle of Hyper-CVAD/MA conditioning without graft-versus-host disease (GVHD) prophylaxis. RESULTS: A total of 31 cycles of microtransplantation were performed. Among them, 6 patients achieved a complete remission (CR) and 2 got partial remission (PR). And the overall response was 8/10. The occurrence of grades III-IV neutrocytopenia and thrombocytopenia was almost 100% after Hyper-CVAD/MA conditioning, but the median recovery times of neutrophils and platelets were 9 days and 14 days respectively because of programmed infusions of G-PBSCs. And 18 bouts of G-PBSC infusion related fever were observed. No GVHD was observed in any of them during treatment. Up to March 2013, 6 patients survived while another 4 died. The 1- and 3-year overall survival and disease-free survival rates were the same (60%). CONCLUSION: The novel therapy of microtransplantation may improve outcome and avoid GVHD in patients with relapsing or refractory lymphoma.

[Analysis on Cellular Immune Function Recovery after HLA Mismatched Allogeneic Hematopoietic Stem Cell Micro-Transp- lantation in Elderly AML Patients].

OBJECTIVE: To investigate the recovery of cellular immunity in elderly patients with acute myeloid leukemia (AML) after micro-transplantation (MST) and the changes of cellular immunity during relapse, as well as their clinical significance. METHODS: A total of 41 elderly AML patients who received MST treatment in a single center and 25 healthy elderly people were included. Immune function among different age groups in normal population was compared. Furthermore, immune fuction was compared between elderly AML patients of different age groups who achieved continuous complete remission (CR) after MST treatment and normal controls, between high risk group and medium-low risk group, as well as among before diagnosis, after CR, and relapse. Peripheral blood of patients and normal controls was collected, and the percentage of lymphocyte subsets was detected by multi-color flow cytometry. RESULTS: Thirty-five patients achieved CR after MST treatment while six patients did not. After MST treatment, CD3+ T cells, CD8+T cells and activated T cells in all age groups were higher than normal. Significant recovery of CD3+ and CD8+T cells was observed in both high risk and medium-low risk groups, and the overall recovery of immune cells in medium-low risk group was better. It was also observed that B lymphocytes and NK cells could not return to normal levels within 1 year after MST treatment. The proportion of CD3+ T cells, CD4+ T cells, and CD4/CD8 ratio were significantly decreased during relapse compared with continuous CR after MST (P<0.05). CONCLUSION: MST treatment can promote the recovery of CD3+T cells, CD8+T cells and other killer cells, so as to improve the cellular immune function of elderly patients, which provides a new immune cell therapy for elderly AML.

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.

[Efficacy and Prognostic Factors of Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation for Patients with Acute Myeloid Leukemia].

OBJECTIVE: To retrospectively analyze the efficacy and complications of our institution's modified nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST) in treating intermediate-risk acute myeloid leukemia (AML) - first complete remission (CR1) and prognostic factors. METHODS: Clinical data of 50 intermediate-risk AML-CR1 patients who underwent matched related NST at the Fifth Medical Center of Chinese People's Liberation Army General Hospital from August 2004 to April 2021 were collected, the hematopoietic recovery, donor engraftment and complications were observed, and overall survival (OS) rate, leukemia-free survival (LFS) rate, treatment-related mortality (TRM), and cumulative relapse rate were calculated. Statistical analysis of factors affecting prognosis was also preformed. RESULTS: The median times for neutrophil and platelet recovery after transplantation were 10 (6-16) and 13 (6-33) days, respectively. One month after transplantation, 22 patients (44%) achieved full donor chimerism (FDC), and 22 patients (44%) achieved mixed chimerism (MC), among whom 18 cases gradually transited to FDC during 1-11 months, 4 cases maintained MC status. The overall incidence of acute graft-versus-host disease (aGVHD) was 36%, with a rate of 18% for grade II-IV aGVHD and a median onset time of 45 (20-70) days after transplantation. The overall incidence of chronic GVHD (cGVHD) was 34%, with 20% and 14% of patients having limited or extensive cGVHD, respectively. The incidence rates of infections, interstitial pneumonia, and hemorrhagic cystitis were 30%, 10%, and 16%, respectively. The 5-year OS rate, LFS rate, TRM, and cumulative relapse rate were 68%, 64%, 16%, and 20%, respectively. The increase of the number of CD34+ cells infused had shortened the recovery time for neutrophils and platelets (r =0.563, r =0.350). The number of CD34+ cells infused significantly influenced the occurrence of extensive cGVHD (OR =1.36, 95%CI : 1.06-1.84, P =0.024). CONCLUSION: Modified NST is effective in treating intermediate-risk AML-CR1 patients, however, further expansion of sample size is needed to study prognostic factors.

Mechanisms of Thymic Repair of In Vitro-Induced Precursor T Cells as a Haploidentical Hematopoietic Stem Cell Transplantation Regimen.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is currently an effective treatment for malignant hematologic disease, but the immune deficiency and severe infection triggered by slow immune reconstitution are the main causes of high mortality and transplant failure. One of these outstanding problems is thymus damage, which is associated with graft-versus-host disease (GVHD), and preconditioning including irradiation and chemotherapy. Therefore, rapid repair of damaged thymus and rapid proliferation of thymus-derived donor T cells post-transplantation are key to solving the problem. This study was designed to accelerate the recovery of thymus-derived T cells post-transplantation. Wild-type mice with normal immunity were used as recipients in a haplo-HSCT mouse model to mimic clinical haplo-HSCT. A modified cell culture system using Notch ligand Delta4 and IL-7 was established that is capable of inducing and amplifying the differentiation and proliferation of hematopoietic stem cells into precursor T (preT) cells in vitro. The haplo-HSCT protocol included preT and G-CSF-mobilized donor splenic mononuclear cell (MNC) coinfusion in one group and MNC infusion alone in a second group. Thymic GVHD, thymic repair, and thymus-derived T cell development were compared in the 2 groups by polychromatic immunofluorescence tracking, flow cytometry, and detection of T cell receptor Vß. The thymus homing and T cell regeneration of allogenic preT cells were observed. The functions of preT cells in accelerating immune reconstitution, restoring thymic architecture, weakening graft-versus-host (GVH) effects, and enhancing immunotolerance post-transplantation were demonstrated. Further studies revealed that allogeneic preT cells induced by a culture system containing IL-7 and Delta4 highly express ccr9 and RANKL. Interestingly, RANK expression was promoted after preT cell thymus homing. These results suggest that the RANK/RANKL pathway may play an important role in thymus homing. Our results provide a potential therapeutic option to optimize haplo-HSCT, and also open up a new field of T cell therapy for artificial induction of allogeneic preT cells in vitro to repair the damaged thymus from irradiation and chemotherapy and to compensate for the recovery of immune function in patients with immune deficiency of various etiologies.

HLA-mismatched allogeneic adoptive immune therapy in severely immunosuppressed AIDS patients.

Severely immunosuppressed AIDS patients with recurrent opportunistic infections (OIs) represent an unmet medical need even in the era of antiretroviral therapy (ART). Here we report the development of a human leukocyte antigen (HLA)-mismatched allogeneic adaptive immune therapy (AAIT) for severely immunosuppressed AIDS patients. Twelve severely immunosuppressed AIDS patients with severe OIs were enrolled in this single-arm study. Qualified donors received subcutaneous recombinant granulocyte-colony-stimulating factor twice daily for 4-5 days to stimulate hematopoiesis. Peripheral blood mononuclear cells were collected from these donors via leukapheresis and transfused into the coupled patients. Clinical, immunological, and virological parameters were monitored during a 12-month follow-up period. We found AAIT combined with ART was safe and well-tolerated at the examined doses and transfusion regimen in all 12 patients. Improvements in clinical symptoms were evident throughout the study period. All patients exhibited a steady increase of peripheral CD4+ T cells from a median 10.5 to 207.5 cells/µl. Rapid increase in peripheral CD8+ T-cell count from a median 416.5 to 1206.5 cells/µl was found in the first 90 days since initiation of AAIT. In addition, their inflammatory cytokine levels and HIV RNA viral load decreased. A short-term microchimerism with donor cells was found. There were no adverse events associated with graft-versus-host disease throughout the study period. Overall, AAIT treatment was safe, and might help severely immunosuppressed AIDS patients to achieve a better immune restoration. A further clinical trial with control is necessary to confirm the efficacy of AAIT medication.

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.

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.

Do We Need Full Donor Chimerism? How Alloreactive Cell Therapies without Substantial Engraftment Might Treat Hematologic Cancers.

"Alloreactive cell therapy without substantial engraftment"; (ACT-WiSE) refers to adoptive transfer of natural ("non-engineered") human leukocyte antigen-mismatched lymphocytes to mediate anti-neoplastic alloreactivity in recipients without employing pharmacologic immunosuppression. By definition, ACT-WiSE entails subsequent rejection of most, if not all, donor cells. Macrochimerism is transient and microchimerism may be either short-lived or persistent. This strategy harnesses the anticancer potency of alloreactivity without incurring significant risk of graft-versus-host disease. "Microtransplantation" refers to a form of ACT-WiSE where the donor cell product contains hematopoietic progenitor cells. Microtransplantation therefore accelerates hematopoietic recovery and its immunomodulatory effects may differ from other forms of ACT-WiSE. Recent studies suggest that various forms of ACT-WiSE, including microtransplantation, may improve chemosensitivity in patients with myeloid malignancies, resulting in higher complete remission rates and increased survival. Microtransplantation has also demonstrated promising pilot results in relapsed or refractory Non-Hodgkin and Hodgkin lymphoma. ACT-WiSE and microtransplantation may establish a new class of allogeneic cell therapy of particular relevance to persons not considered candidates for traditional allogeneic hematopoietic cell transplantation (AHCT). Open questions include the optimal timing and cell dose of ACT-WiSE, which donor factors contribute to efficacy, and whether these remissions are durable after eradication of donor cells. We extrapolate from lessons learned in the course of traditional and haploidentical AHCT to propose ways of optimizing ACT-WiSE. We divide these into donor-related strategies (including rational donor selection and boosting NK-cell and T-cell alloreactivity) and patient- related strategies (that may favor development of autologous NK-cell and T-cell mediated anticancer cytotoxicity in the post-ACT-WiSE window).

[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.

[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.

[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.

[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.