Granulocyte Colony-stimulating Factor-mobilized Peripheral Blood Research Articles

Allogeneic transplantation of bone marrow versus peripheral blood stem cells from HLA-identical relatives in patients with myelodysplastic syndromes and oligoblastic acute myeloid leukemia: a propensity score analysis of a nationwide database.

Bone marrow (BM) and granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (PBSC) are used as grafts from HLA-identical-related donors for adults with myelodysplastic syndrome (MDS). To assess the impact of graft sources on post-transplant outcomes in MDS patients, we conducted a retrospective analysis of a nationwide database. A total of 247 and 280 patients underwent transplantation with BM and PBSC, respectively. The inverse probability of treatment weighting (IPTW) methods revealed that overall survival (OS) was comparable between BM and PBSC (P = .129), but PBSC transplantation was associated with worse graft-versus-host disease (GVHD)-free/relapse-free survival (GRFS) (hazard rate [HR], 1.24; 95% confidence intervals [CIs], 1.00-1.53; P = 0.049) and chronic GVHD-free and relapse-free survival (CRFS) (HR, 1.29; 95% CIs, 1.13-1.73; P = 0.002) than BM transplantation. In the propensity score matched cohort (BM, n = 216; PBSC, n = 216), no significant differences were observed in OS and relapse; 3-year OS rates were 64.7% and 60.0% (P = 0.107), while 3-year relapse rates were 27.1% and 23.5% (P = 0.255) in BM and PBSC, respectively. Three-year GRFS rates (36.6% vs. 29.2%; P = 0.006), CRFS rate (37.7% vs. 32.5%; P = 0.003), and non-relapse mortality rates (13.9% vs. 21.1%; P = 0.020) were better in BM than in PBSC. The present study showed that BM transplantation provides a comparable survival benefit with PBSC transplantation and did not identify an enhanced graft-versus-MDS effect to reduce the incidence of relapse in PBSC transplantation.

Reduced GvHD in Recipients of BM Derived Versus G-CSF Mobilized Plasmacytoid Dendritic Cells: Role of Inducible IL-12

Introduction: BMT CTN 0201, a randomized clinical trial, demonstrated that overall survival is equivalent between recipients of allogeneic bone marrow (BM) vs. granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood grafts. Increased graft failure was observed in recipients of BM grafts, while recipients of G-CSF mobilized peripheral blood (G-PB) grafts had more chronic graft-versus-host disease (GvHD) (Anasetti NEJM 2012). Donor plasmacytoid dendritic cell (pDC) content is associated with increased overall survival and decreased acute GvHD related mortality among recipients of BM grafts from unrelated donors, but not G-PB grafts (Waller JCO 2014). We have previously shown that in murine bone marrow transplant (BMT), donor pDC from the bone marrow reduce GvHD and improve survival while maintaining graft-versus-leukemia effect when added to grafts containing purified hematopoietic stem cells (HSC) and T cells (Lu BLOOD 2012). We hypothesized that the difference in chronic GvHD observed in BMT CTN 0201 is due to donor pDC content and function that vary according to graft source differences: BM vs. G-PB.Methods and Results: To assess the effects of pDC source on overall survival and GvHD, we used an established MHC mismatched model of murine BMT (C57BL/6 to B10.BR). To test whether the graft source of donor pDC was relevant to GvHD, we used flow cytometry to sort populations of HSC and T cells from untreated donor mice and pDC from either untreated BM or G-CSF mobilized splenocytes. We performed a BMT with 5,000 HSC, 1 million T cells, and either 50,000 BM or G-PB pDC from C57BL/6 mice into B10.BR recipients. Overall survival was significantly reduced among recipients of G-PB donor pDC compared with recipients of BM pDC, while late stage GvHD was higher in recipients of G-PB pDC (Fig. 1).To determine possible differences in function of pDC from each graft source: BM vs. G-PB, we assessed cytokine and enzymatic profiles by flow cytometry. Intracellular levels of IL-12 was significantly higher in CpG stimulate purified BM pDC compared to G-PB pDC co-cultured with purified T cells stimulated by anti-CD3/CD28 beads (Fig. 2). Additionally, Th1 polarization was higher in T cells sitmulated with anti-CD3/CD38 and cocultured with BM pDC, resulting in higher TNFa and IFNy production from these T cells. Levels of intracellular cytokines IFNa and IL-10 and levels of IDO showed no significant differences between pDC from Bm vs. G-PB.The transplant detailed above was repeated with BM pDC from wild-type (WT) mice or IL-12 KO mice. Overall survival of recipients of IL-12 KO BM pDC was significantly reduced when compared with recipients of WT BM pDC (Fig. 3).Conclusion: These data confirm that donor BM pDC are protective against lethal GvHD and indicate a loss of GvHD protection in donor pDC from G-PB grafts due to decreased inducible IL-12 synthesis. Because both graft sources are widely used clinically, mechanisms to recapitulate the immune-modulating properties of BM pDC in pDC of G-PB grafts are of great interest to the BMT field. [Display omitted] DisclosuresWaller:National Institutes of Health: Research Funding; Celldex: Consultancy; Cerus: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Katz Foundation: Research Funding; AMGEN: Consultancy; Chimerix: Equity Ownership; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; PRA: Consultancy; Coulter Foundation: Research Funding.

HLA-Haploidentical Hematopoietic Cell Transplantation for Treatment of Nonmalignant Diseases Using Nonmyeloablative Conditioning and Post-Transplant Cyclophosphamide.

Allogeneic hematopoietic cell transplant (HCT) is often the only curative therapy for patients with nonmalignant diseases; however, many patients do not have an HLA-matched donor. Historically, poor survival has been seen after HLA-haploidentical HCT because of poor immune reconstitution, increased infections, graft-versus-host disease (GVHD), and graft failure. Encouraging results have been reported using a nonmyeloablative T cell-replete HLA-haploidentical transplant approach in patients with hematologic malignancies. Here we report the outcomes of 23 patients with various nonmalignant diseases using a similar approach. Patients received HLA-haploidentical bone marrow (n=17) or granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (n=6) grafts after conditioning with cyclophosphamide 50 mg/kg, fludarabine 150 mg/m2, and 2 or 4 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclophosphamide, mycophenolate mofetil, tacrolimus, ± sirolimus. Median patient age at HCT was 10.8 years. Day 100 transplant-related mortality (TRM) was 0%. Two patients died at later time points, 1 from intracranial hemorrhage/disseminated fungal infection in the setting of graft failure and 1 from infection/GVHD. The estimated probabilities of grades II to IV and III to IV acute GVHD at day 100 and 2-year National Institutes of Health consensus chronic GVHD were 78%, 26%, and 42%, respectively. With a median follow-up of 2.5 years, the 2-year overall and event-free rates of survival were 91% and 78%, respectively. These results are encouraging and demonstrate favorable disease-specific lineage engraftment with low TRM in patients with nonmalignant diseases using nonmyeloablative conditioning followed by T cell-replete HLA-haploidentical grafts. However, additional strategies are needed for GVHD prevention to make this a viable treatment approach for patients with nonmalignant diseases.

Nonmyeloablative TLI-ATG conditioning for allogeneic transplantation: mature follow-up from a large single-center cohort.

Nonmyeloablative total lymphoid irradiation and antithymocyte globulin (TLI-ATG) conditioning is protective against graft-versus-host disease (GVHD), while retaining graft-versus-tumor activity across various hematologic malignancies. We report our comprehensive experience using TLI-ATG conditioning in 612 patients with hematologic malignancies who underwent allogeneic transplantation at Stanford University from 2001 to 2016. All patients received granulocyte colony-stimulating factor-mobilized peripheral blood grafts and cyclosporine and mycophenolate mofetil for GVHD prophylaxis. The median age was 60 years (range, 21-78), with a median follow-up of 6.0 years (range, 1.0-16.4). Common diagnoses included acute myeloid leukemia (AML; n = 193), myelodysplastic syndrome (MDS; n = 94), chronic lymphocytic leukemia (CLL; n = 80), non-Hodgkin lymphoma (NHL; n = 175), and Hodgkin lymphoma (HL; n = 35). Thirty-four percent of patients had a comorbidity index ≥3, 30% had a high to very high disease risk index, and 56% received unrelated donor grafts, including 15% with HLA-mismatched donors. Ninety-eight percent underwent transplant in the outpatient setting, and 57% were never hospitalized from days 0 through 100. The 1-year rates of nonrelapse mortality (NRM), grade II-IV acute GVHD, and extensive chronic GVHD were 9%, 14%, and 22%, respectively. The 4-year estimates for overall and progression-free survival were 42% and 32% for AML, 30% and 21% for MDS, 67% and 43% for CLL, 68% and 45% for NHL, and 78% and 49% for HL. Mixed chimerism correlated with the risk of relapse. TLI-ATG conditioning was well tolerated, with low rates of GVHD and NRM. Durable remissions were observed across hematologic malignancies, with particularly favorable outcomes for heavily pretreated lymphomas. Several efforts are underway to augment donor chimerism and reduce relapse rates while maintaining the favorable safety and tolerability profile of this regimen.

Impact of Body Mass Index on Outcomes of Hematopoietic Stem Cell Transplantation in Adults

This retrospective analysis of 2503 adult (age ≥20 years) allogeneic hematopoietic cell transplantation (HCT) recipients assessed the effect of body mass index (BMI) on transplantation outcomes. The median patient age was 51.7years. Patients with both nonmalignant and malignant diagnoses were included. Patients received either a myeloablative (52%) or a reduced-intensity (48%) conditioning regimen. Donors were either related (42%) or unrelated (58%). Cord blood recipients were excluded. Granulocyte colony-stimulating factor-mobilized peripheral blood cells were the stem cell source in 86% of transplantations. Graft-versus-host disease prophylaxis included at least 2 immunosuppressive agents, 1 of which was a calcineurin inhibitor. Patient groups were categorized as underweight, normal weight, overweight, obese, or very obese based on BMI. Endpoints included day +100 mortality, overall mortality, nonrelapse mortality (NRM), and relapse. Changes in nutritional status, based on laboratory parameters, were also examined. Underweight patients had significantly lower early and overall survival and greater NRM. Very obese patients had increased NRM, which was associated with the intensity of conditioning regimen. With long-term follow-up, increasing NRM was associated with both underweight and obese patients compared with normal-weight individuals. Changes in serum protein and albumin levels did not correlate with BMI. Although enteral nutrition is now recommended for some undernourished patients, the efficacy of enteral or parenteral nutrition has not been well studied. For obese patients, there are no guidelines regarding weight loss before transplantation, and acute weight loss in the pretransplantation period may be detrimental.

Preventing relapse after haematopoietic stem cell transplantation for acute leukaemia: the role of post-transplantation minimal residual disease (MRD) monitoring and MRD-directed intervention.

Relapse is the main cause of treatment failure after allogeneic haematopoietic stem cell transplantation (allo-HSCT) for acute leukaemia (AL). Post-transplantation minimal residual disease (MRD) monitoring enables risk stratification and identifies AL patients at higher risk of relapse. MRD assessment primarily involves the determination of leukaemia-associated immunophenotypic patterns using multiparameter flow cytometry, and the polymerase chain reaction (PCR)-based evaluation of expression levels of leukaemia-related genes (specific reciprocal gene rearrangements and other mutation types). In addition, next generation sequencing and digital PCR may further enrich current MRD detection. Several MRD-directed interventions have demonstrated the ability to reduce the risk of relapse with acceptable treatment-related toxicities. Donor lymphocyte infusion (DLI) is the most important intervention for MRD-positive patients, while several modified strategies, such as granulocyte colony-stimulating factor-mobilized peripheral blood cells followed by short term immune suppression and escalating dose regimen, further improve the safety and efficacy of DLI. Interferon therapy, targeted drugs, and hypomethylating agents have also been introduced for MRD-directed interventions. Referring to the issues of whether and who would benefit from pre-emptive intervention according to MRD, in this review, we summarized this rapidly evolving area of MRD monitoring and MRD-directed interventions in AL patients after allo-HSCT.

Priming mobilized peripheral blood mononuclear cells with the "activated platelet supernatant" enhances the efficacy of cell therapy for myocardial infarction of rats.

Various methods are used to augment the efficacy of cell therapy in myocardial infarction (MI). In this study, we used the "activated platelet supernatant (APS)" to prime autologous "granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells ((mob) PBMCs)" and investigated the efficacy of cell-based therapy in MI. Rat (mob) PBMCs were isolated after daily subcutaneous injections of G-CSF at 100μg/kg for 3days. APS was isolated separately after activating rat platelets with thrombin 0.5U/mL for 2hours. Priming was performed with APS for 6hours. To check the paracrine effect of primed (mob) PBMCs, we used the 36-hour culture supernatant of the primed cells. A rat MI model was used for an in vivo model. Cytokines such as IL-1β, IL-10, and TGFβ were 3.7±0.9-fold, 3.4±1.2-fold, and 1.2±0.1-fold higher in APS, respectively, compared with naïve platelet supernatant. By APS priming, (mob) PBMCs showed M2 polarization and upregulation of angiogenic molecules (i.e., TEK, IL-10, CXCL1, and CX3CR1). APS-primed (mob) PBMCs had a 2.3-fold increased adhesion ability, induced by upregulated integrins. Rat endothelial cells cultured in the 36-hour culture supernatant of APS-primed (mob) PBMCs showed a 1.6-fold augmented proliferation and capillary network formation. In vivo transplantation of APS-primed (mob) PBMCs into rat MI models showed a significant trend of reduction in fibrosis area (P=.001) and wall thinning (P=.030), which lead to improvement in cardiac function measured by echocardiography. Our data reveal that APS priming can enhance the wound-healing potential of (mob) PBMCs. APS priming may be a promising method for cell-based therapy of MI.

Isolation and Characterization of Rat Mesenchymal Stem Cells Derived from Granulocyte Colony-Stimulating Factor-Mobilized Peripheral Blood

Mesenchymal stem cells (MSCs) have been isolated from many tissues and organs. However, there is much dispute as to whether MSCs exist in peripheral blood. This may be due to the limited identification methods of MSCs, especially the lack of detection markers for phenotypic characteristics. In this study, as many as 10 surface markers of MSCs derived from rat peripheral blood (rPBMSCs) were analyzed after granulocyte colony-stimulating factor mobilization. Our results suggest that mobilized rPBMSCs overexpress mesenchymal markers, including CD90, CD44, CD29, CD73 and CD105, but do not express CD45, CD11b, CD79a, CD34 or HLA-DR. This is in conformity with the standard definition of MSCs by the International Society for Cellular Therapy. In addition, the colony-forming efficiency of the mobilized rat peripheral blood was 15.83 ± 1.61/10⁶, significantly outnumbering that of the nonmobilized group, which was 0.28 ± 0.1/10⁶ (p < 0.01). Combining the growth characteristics with the differential capacities of mobilized rPBMSCs towards forming osteocytes, chondrocytes and adipocytes, we further confirmed the existence of rPBMSCs. Additionally, this treatment could improve locomotive function after spinal cord injury (SCI) in rats. Due to their convenient collection, fewer complications, cost effectiveness and suitability for autograft, PBMSCs might be a substitute for MSCs derived from bone marrow and provide promising prospects for the cell-based therapy of SCI.

Defining the optimal plasmacytoid dendritic cell graft source for hematopoietic stem cell transplantation

Abstract Purpose Allogeneic hematopoietic stem cell transplantation (HSCT) can cure patients with blood disorders and malignancies. Stem cell source can be bone marrow (BM) or peripheral blood mobilized by granulocyte-colony stimulating factor (G-CSF). Plasmacytoid dendritic cells (pDC) reduce the incidence and severity of GvHD. The BMT CTN 0201 study indicates overall survival, relapse, and the incidence of acute GvHD are similar in patients that receive BM versus G-CSF mobilized peripheral blood (G-PB) grafts, but increased incidence of chronic GvHD in patients who receive G-PB grafts. To explore the effect of graft source on the functional properties of pDC, we compared the effect of G-CSF mobilized pDC to BM pDC in the regulation of GvHD in a murine allogeneic HSCT model. Methods 1) Expression of surface markers on BM and G-CSF treated pDC were assessed by flow cytometry. 2) We performed an HSCT from C57BL/6 mice into B10.BR recipients. Groups include HSC only, HSC + T cells, and HSC + T cells + BM pDC or G-CSF mobilized pDC. Hematopoietic chimerism, GvHD, and overall survival were assessed. Results 1) BM pDC express CCR9 (marker for tolerogenic pDC) and CD62L at a higher frequency, suggesting enhanced homing to the gut, thymus, and lymph nodes. G-CSF mobilized pDC express maturational markers MHC II and CD86 at a higher frequency, suggesting effectiveness in activating T cells. 2) Mice that received transplants consisting of only HSC rejected the transplant. Mice that received transplants with T cells had greater than 90% donor hematopoietic cells. GvHD more than 40 days post transplant was highest in mice that received G-CSF mobilized pDC. Survival without pDC was under 60%. Groups that received pDC had an overall survival of greater than 80%.

A Study of Human Leukocyte Antigen Mismatched Cellular Therapy (Stem Cell Microtransplantation) in High-Risk Myelodysplastic Syndrome or Transformed Acute Myelogenous Leukemia

The treatment outcomes of myelodysplastic syndrome (MDS) and transformed acute myelogenous leukemia (tAML) remain very unsatisfactory. We designed a combination of human leukocyte antigen (HLA)-mismatched hematopoietic stem cell microtransplantation (MST) with chemotherapy for patients with MDS and tAML and evaluated its effects and toxicity. Patients were between 13 and 79 years old. Patients with MDS (n=21) were given HLA-mismatched MST combined with decitabine and cytarabine; patients with tAML (n=22) were given HLA-mismatched MST combined with decitabine and cytarabine, and also mitoxantrone. Patients in complete remission (CR) also received MST plus decitabine and medium-dose cytarabine chemotherapy without graft-versus-host disease (GVHD) prophylaxis. The overall response rate of the patients with MDS was significantly higher than that of those with tAML (81% vs. 50%; p=.03). The CR rates were 52.4% and 36.4% in the two groups, respectively. There was no difference in the cytogenetic CR rate between the MDS and tAML groups (85.7% vs. 70%, respectively; p=.7). The 24-month overall survival of the patients with MDS was significantly higher than that of the patients with tAML (84.7% and 34.1%, respectively; p=.003). The median recovery times of neutrophils and platelets were, respectively, 14 and 17 days in the patients with MDS, and 16 and 19 days in those with tAML. The treatment-related mortality rates were 4.8% and 18.2%, respectively, in the MDS and tAML groups (p=.34). No GVHD was observed in any patient. Microtransplantation combined with decitabine and chemotherapy may provide a novel, effective, and safe treatment for high-risk MDS and tAML. Microtransplantation (MST) refers to regular chemotherapy combined with granulocyte colony-stimulating factor-mobilized peripheral blood stem cell infusion of human leukocyte antigen-mismatched donor cells without using immunosuppressive agents. It aims to support hematopoietic recovery and perform graft-versus-leukemia (GVL) effects but differs from traditional allogeneic stem cell transplantation because the rate of donor cell chimerism is low and there is and no graft-versus-host disease (GVHD) risk. Thus, a trial was designed to evaluate the safety and efficacy of MST in patients with myelodysplastic syndrome and those with transformed acute myelogenous leukemia. Higher complete remission and cytogenetic complete response rates were observed, and the treatment improved disease progress-free survival, sped hematopoietic recovery, and avoided GVHD.

A Phase II Study Evaluating the Safety and Efficacy of Subcutaneous Plerixafor for the Mobilization and Transplantation of HLA-Matched Sibling Donor Hematopoietic Stem Cells in Recipients with Hematological Malignancies

▪The optimal source of donor hematopoietic stem cells (HSC) is controversial. Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood (G-PB) has replaced bone marrow (BM) as the most common allograft source in adults but is associated with donor morbidity and higher rates of chronic graft versus host disease (GVHD) compared to BM. The CXCR4 antagonist plerixafor (Px) mobilizes HSC into the PB (Px-PB) faster than G-CSF and preliminary data suggest both quantitative and qualitative differences in allograft content that may impact clinical outcomes. We sought to assess the efficacy and safety of transplanted allografts collected following mobilization with Px alone in HLA-identical sibling transplantation. This was a Phase II, two-strata, multi-center prospective trial (NCT01696461) to evaluate Px-PB allografts prior to reduced intensity conditioning (RIC) and myeloablative conditioning (MAC) based hematopoietic cell transplantation (HCT). Patients aged 18-65 years with an HLA-ID sibling donor and a hematological malignancy suitable for HCT were eligible. The primary objective was to determine the proportion of donors whose cells could be successfully mobilized and collected with a sufficient CD34+ cell dose using Px as the sole mobilizing agent. Px mobilization was considered successful if ≥ 2.0x10^6 CD34+ cells/kg recipient weight were collected in no more than two leukapheresis (LP) collections. All donors receiving Px were included in the analysis of the primary objective based on the intention-to-treat principle. Secondary objectives included the incidence of acute and chronic adverse events in donors, rates of hematopoietic engraftment, donor chimerism, rates of acute and chronic GVHD, non-relapse mortality (NRM), progression free survival (PFS) and overall survival (OS) for the recipients. From July 2013 to December 2014, 64 donor/recipient pairs were enrolled at 12 centers. Donors received Px at 240μg/kg subcutaneously 4 hours prior to LP. LP was performed processing at least 4X blood volume for up to two consecutive days (a third day was allowed for low CD34+ cell yields after 2 LP procedures) to achieve a target CD34+ cell dose of ≥ 4.0 x 10^6/kg recipient weight with a minimum goal of ≥ 2.0 x 10^6/kg. All allografts were cryopreserved. GVHD prophylaxis included cyclosporine or tacrolimus in combination with methotrexate, mycophenolate mofetil, or sirolimus. G-CSF was given routinely post HCT only to MAC recipients. Patient demographics are provided in Table 1. The median donor age was 56 years (18-65). 64% of the donors were male. Donors underwent one (23%), two (72%), or three (5%) LP procedures. 63 of 64 (98%) donors achieved the primary objective. The median total CD34+ cell dose/kg recipient weight collected within 2 days was 4.6 (0.9-9.6). Maximal donor toxicity following Px injection and LP was grades 0 (30%), 1 (52%), 2 (17%), and 3 (2%). Bloating, flatulence, abdominal pain, headache, paresthesisas, injection site reaction, and dizziness were the most commonly observed toxicities. Bone pain was not observed. The one grade 3 toxicity was a vasovagal episode felt related to LP and unlikely to Px. Toxicities typically resolved within a week of LP. The median follow up is 6.3 months. Median days to ANC (>0.5 x10^9/L) and Platelet count (>20 x 10^9/L) recovery were 13.5 (10-148) and 19 (1-76) after MAC and 14.5 (0-25) and 18 (0-141) after RIC, respectively. The cumulative incidence of acute GVHD grades 2-4 and 3-4 at day 100 were 47% (95% CI: 30-64) and 9% (95% CI: 2-22) after MAC and 19% (95% CI: 6-38) and 5% (95% CI: 0-18) after RIC. Probability of NRM at day 100 was 4% (95% CI: 0-13) and 0% after MAC and RIC, respectively. The probability of OS at day 100 was 97% (95% CI: 88-100) and 90% (95% CI: 78-98) after MAC and RIC, respectively. In conclusion, this is the first multi-center trial to demonstrate that as an alternative to G-CSF, Plerixafor rapidly, safely, and effectively mobilizes sufficient numbers of CD34+ cells from HLA-ID sibling donors for HCT following both RIC and MAC regimens. Engraftment was generally prompt and early results of secondary endpoints in recipients are encouraging. Longer follow-up and more extensive analysis of donor allografts and recipient outcomes will be presented at the time of the meeting. Research support was provided in part by Genzyme, a Sanofi Company. [Display omitted] DisclosuresChen:Bayer: Consultancy, Research Funding. Devine:Genzyme: Research Funding.

Negative Impact of Unidirectional Host-versus-Graft Killer Cell Immunoglobulin-like Receptor Ligand Mismatch on Transplantation Outcomes after Unmanipulated Haploidentical Peripheral Blood Stem Cell Transplantation for Acute Myeloid Leukemia.

This study explored the influence of mismatched inhibitory killer cell immunoglobulin-like receptor (KIR) ligands on the outcome of haploidentical transplantation using T cell-replete, granulocyte colony-stimulating factor-mobilized peripheral blood stem cells in adult patients with acute myeloid leukemia (AML). Three groups were examined: unidirectional graft-versus-host KIR ligand mismatched (GVH-KIR-MM; n= 33), bidirectional KIR ligand matched (KIR-M; n= 41), and unidirectional host-versus-graft KIR ligand mismatched (HVG-KIR-MM; n= 26). All recipients were treated with the same conditioning regimen (800 cGy total body irradiation, fludarabine, busulfan, and antithymocyte globulin). After a median follow-up of 26 months, the 2-year cumulative incidence of relapse was significantly higher in HVG-KIR-MM (40.3% ± 10.3%) versus others (18.9% ± 4.8%, P= .044). In the standard-risk group, the 2-year disease-free survival (DFS) was significantly lower in HVG-KIR-MM (51.8% ± 11.2%) compared with GVH-KIR-MM (88% ± 8.1%, P= .025). Multivariate analysis showed that HVG-KIR-MM was significantly associated with higher relapse (hazard ratio [HR], 10.7; P= .002) and lower DFS (HR, 3.4; P= .012). Subgroup analysis revealed increased DFS with higher doses of CD3(+)CD8(+) and CD3(-)CD56(+) grafts in GVH-KIR-MM (90.9% ± 8.7%, P= .006); there was no such effect in the other groups. Although our conclusions are limited by the absence of donor KIR genotype data, our study suggests unidirectional KIR ligand incompatibility in the host-versus-graft vector has a detrimental effect on T cell-replete haploidentical transplantation outcomes in adult patients with AML.

Superior Potential of CD34-Positive Cells Compared to Total Mononuclear Cells for Healing of Nonunion Following Bone Fracture.

We recently demonstrated that the local transplantation of human peripheral blood (PB) CD34(+) cells, an endothelial/hematopoietic progenitor cell-rich population, contributes to fracture repair via vasculogenesis/angiogenesis and osteogenesis. Human PB mononuclear cells (MNCs) are also considered a potential cell fraction for neovascularization. We have previously shown the feasibility of human PB MNCs to enhance fracture healing. However, there is no report directly comparing the efficacy for fracture repair between CD34(+) cells and MNCs. In addition, an unhealing fracture model, which does not accurately resemble a clinical setting, was used in our previous studies. To overcome these issues, we compared the capacity of human granulocyte colony-stimulating factor-mobilized PB (GM-PB) CD34(+) cells and human GM-PB MNCs in a nonunion model, which more closely resembles a clinical setting. First, the effect of local transplantation of 1 × 10(5) GM-PB CD34(+) cells (CD34(+) group), 1 × 10(7) GM-PB MNCs (containing approximately 1 × 10(5) GM-PB CD34(+) cells) (MNC group), and phosphate-buffered saline (PBS) (PBS group) on nonunion healing was compared. Similar augmentation of blood flow recovery at perinonunion sites was observed in the CD34(+) and MNC groups. Meanwhile, a superior effect on nonunion repair was revealed by radiological, histological, and functional assessment in the CD34(+) group compared with the other groups. Moreover, through in vivo and in vitro experiments, excessive inflammation induced by GM-PB MNCs was confirmed and believed to be one of the mechanisms underlying this potency difference. These results strongly suggest that local transplantation of GM-PB CD34(+) cells is a practical and effective strategy for treatment of nonunion after fracture.

Mononuclear cells from the cord blood and granulocytecolony stimulating factor-mobilized peripheral blood: is there a potential for treatment of cerebral palsy?

To investigate a possible therapeutic mechanism of cell therapy in the field of cerebral palsy using granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (mPBMCs), we compared the expression of inflammatory cytokines and neurotrophic factors in PBMCs and mPBMCs from children with cerebral palsy to those from healthy adult donors and to cord blood mononuclear cells donated from healthy newborns. No significant differences in expression of neurotrophic factors were found between PBMCs and mPBMCs. However, in cerebral palsy children, the expression of interleukin-6 was significantly increased in mPBMCs as compared to PBMCs, and the expression of interleukin-3 was significantly decreased in mPBMCs as compared to PBMCs. In healthy adults, the expression levels of both interleukin-1β and interleukin-6 were significantly increased in mPBMCs as compared to PBMCs. The expression of brain-derived neurotrophic factors in mPBMC from cerebral palsy children was significantly higher than that in the cord blood or mPBMCs from healthy adults. The expression of G-CSF in mPBMCs from cerebral palsy children was comparable to that in the cord blood but significantly higher than that in mPBMCs from healthy adults. Lower expression of pro-inflammatory cytokines (interleukin-1β, interleukin-3, and -6) and higher expression of anti-inflammatory cytokines (interleukin-8 and interleukin-9) were observed from the cord blood and mPBMCs from cerebral palsy children rather than from healthy adults. These findings indicate that mPBMCs from cerebral palsy and cord blood mononuclear cells from healthy newborns have the potential to become seed cells for treatment of cerebral palsy.

Quantitative polymerase chain reaction for detection of human herpesvirus-7 infection in umbilical cord blood donors.

Umbilical cord blood (UCB) has been a reasonable alternative to granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow, as a source of hematopoietic stem cells with a lower risk of graft-versus-host disease. In immunocompromised hosts after transplantation, the risk of viral infection in adults, especially with beta-herpesviruses such as human herpesvirus-7 (HHV-7), may be increased. This virus in immunocompromised patients can be reactivated from latency and converted to an active phase. Therefore, light-upon-extension real-time polymerase chain reaction (PCR) was developed to assess the prevalence and load of HHV-7 in the plasma and buffy coat of donors. About 825 UCB samples under standard protocol from donors were collected. Then, DNA from plasma and buffy coat was extracted and quantitative real-time PCR was performed with light-upon-extension primers. Overall, HHV-7 was detected in 3.64% (30/825) of UCB donors. HHV-7 DNA was detected in 26 (3.2%) buffy coat samples (latent infection), and only 4 (0.48%) of them were positive for HHV-7 DNA in plasma samples (active infection); the mean HHV-7 viral load was 1.31×10(1) copies/mL in latent infection, and 1.94×10(5) copies/mL in active infection. We suggest that real-time PCR in plasma and buffy coat could be a useful method to detect active and latent HHV-7 infection in UCB donors and determine its role in subsequent transmission events.

Improved survival after transplantation of more donor plasmacytoid dendritic or naïve T cells from unrelated-donor marrow grafts: results from BMTCTN 0201.

To characterize relationships between specific immune cell subsets in bone marrow (BM) or granulocyte colony-stimulating factor-mobilized peripheral blood (PB) stem cells collected from unrelated donors and clinical outcomes of patients undergoing transplantation in BMTCTN 0201. Fresh aliquots of 161 BM and 147 PB stem-cell allografts from North American donors randomly assigned to donate BM or PB stem cells and numbers of transplanted cells were correlated with overall survival (OS), relapse, and graft-versus-host disease (GvHD). Patients with evaluable grafts were similar to all BMTCTN 0201 patients. The numbers of plasmacytoid dendritic cells (pDCs) and naïve T cells (Tns) in BM allografts were independently associated with OS in multivariable analyses including recipient and donor characteristics, such as human leukocyte antigen mismatch, age, and use of antithymocyte globulin. BM recipients of > median number of pDCs, naïve CD8(+) T cells (CD8Tns), or naïve CD4(+) T cells (CD4Tns) had better 3-year OS (pDCs, 56% v 35%; P = .025; CD8Tns, 56% v 37%; P = .012; CD4Tns, 55% v 37%; P = .009). Transplantation of more BM Tns was associated with less grade 3 to 4 acute GvHD but similar rates of relapse. Transplantation of more BM pDCs was associated with fewer deaths resulting from GvHD or from graft rejection. Analysis of PB grafts did not identify a donor cell subset significantly associated with OS, relapse, or GvHD. Donor immune cells in BM but not PB stem-cell grafts were associated with survival after unrelated-donor allogeneic hematopoietic stem-cell transplantation. The biologic activity of donor immune cells in allogeneic transplantation varied between graft sources. Donor grafts with more BM-derived Tns and pDCs favorably regulated post-transplantation immunity in allogeneic hematopoietic stem-cell transplantation.

Endothelial Cell-Selective Adhesion Molecule Marks Human Hematopoietic Stem Cells Regardless Of The HSC Sources

Identification of novel markers associated with hematopoietic stem cells (HSCs) is important to progress basic and clinical research regarding the HSC biology. We previously reported that endothelial cell-selective adhesion molecule (ESAM) marks HSCs throughout life in mice (Yokota et al. Blood, 2009). We also demonstrated that ESAM can be a useful indicator of activated HSCs after bone marrow (BM) injury and that ESAM is functionally important for recovering hematopoiesis by using ESAM knockout mice (Sudo et al. J Immunol, 2012). However, the discrepancy between species has been a long-standing obstacle to apply findings in mice to human. For example, established murine HSC markers such as Sca-1 or CD150 are not expressed on human HSCs. Thus, it is important to know if ESAM marks HSCs beyond species and serves as a functional molecule for the HSC property, but information regarding ESAM expression in human HSCs has been quite limited. In this study, we have examined the ESAM expression pattern on human HSCs derived from diverse sources. In addition, we have performed functional assessment of the ESAM-expressing cells.Cord blood (CB), aspirated BM, and granulocyte-colony stimulating factor-mobilized peripheral blood (GMPB) were obtained from healthy donors. BM was also obtained from head of femora of patients who received the hip replacement surgery. All of the protocols were approved by the Institutional Review Board of Osaka University School of Medicine, and we obtained the written agreement form with informed consent from all participants. Mononuclear cells were separated using Ficoll centrifugation from CB, aspirated BM and GMPB. For preparation of BM cells adjacent to bone tissues, trabecular tissues of femora were treated with 2 mg/ml collagenase IV and DNase and gently agitated for 1 hour at 37 °C. Collected cells were analyzed using flow cytometry for cell surface expression of ESAM and other markers. Further, the CD34+ CD38−cells were fractionated according to the intensity of ESAM expression and evaluated in vivo and in vitro functional assays.Flow cytometry analyses revealed that the majority of CB CD34+ CD38− cells expressed ESAM. According to the expression level, CB CD34+ CD38− cells could be subdivided into three populations, namely ESAM−/Low, ESAMHigh, and ESAMBright. While all CB contained a robust ESAMHigh population in CD34+ CD38− cells, the percentage of ESAMBright cells varied widely among CB samples. The ESAMHigh CD34+ CD38− cells also expressed CD90 and CD133, which are known as HSC markers. Methylcellulose colony-forming assays and limiting dilution assays revealed that ESAMHigh fraction enriches primitive hematopoietic progenitors. Further, ESAMHigh cells also reconstituted the long-term human hematopoiesis in NOD/Shi-scid, IL-2Rγnull (NOG) mice. Therefore, as in mice, ESAMHighmarks authentic HSCs in human.On the other hand, ESAMBright CD34+ CD38− cells showed low colony-forming activities and no reconstitution of human hematopoiesis in NOG mice. These ESAMBright CD34+ CD38− cells expressed CD118/leukemia inhibitor factor receptor and endothelial markers such as VE-Cadherin, Flk-1, and CD146, but not CD45. These results suggested that ESAMBright cells in the CB CD34+ CD38− fraction are non-hematopoietic cells. With respect to the other HSC sources such as aspirated BM and GMPB, almost all CD34+ CD38− cells were ESAMHigh and ESAMBright cells were not found in this fraction. Interestingly, however, ESAMBright cells were found in the CD34+ CD38− fraction isolated from collagenase-treated femora. These BM-derived ESAMBright CD34+ CD38− cells expressed endothelial markers as did the CB-derived cells. They could generate CD31+endothelial cells, but not hematopoietic cells in coculture with MS5 stromal cells with vascular endothelial growth factor, stromal-cell-derived factor, and interleukin 16.In conclusion, ESAM expression serves as a marker to enrich HSCs in human regardless of the HSC sources. In addition, the very high intensity of this marker might be useful to isolate non-hematopoietic progenitors from CD34+ CD38− cells, which has been conventionally used as human HSCs. The common feature of ESAM expression of murine and human HSCs suggests a possibility that functional significance of ESAM expression obtained from mouse studies could be applicable to human. Disclosures:No relevant conflicts of interest to declare.

Donor lymphocyte infusions for relapse after allogeneic transplantation. When, if and for whom?

Donor lymphocyte infusion (DLI) using unstimulated leukapheresis is one of the most effective treatment strategies for patients with hematological malignancies; its graft-versus-leukemia effects make it especially effective in chronic myeloid leukemia patients who relapsed after allogeneic stem cell transplantation (allo-HSCT). However, DLI application is limited by the development of graft-versus-host disease and aplasia, and thus cannot be routinely applied for prophylaxis. Therefore, important questions remain to be answered, such as when, and whom to DLI? Recent advances enable DLI using allografts of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells; allodepleted donor T cells; and infusions of donor-derived, ex vivo-expanded, CD8+ cytotoxic T lymphocyte, which can decrease relapse and improve transplant outcomes. Preemptive immunotherapy of relapse was also introduced based on the determination of mixed chimerism and minimal residual disease. In this review, we summarize the latest developments in recent strategies that will affect future DLI efficacy — focusing on the disadvantages and advantages of each protocol for the treatment, preemptive therapy, and prophylaxis of relapse.

Impact of anti-HLA antibodies on allogeneic hematopoietic stem cell transplantation outcomes after reduced-intensity conditioning regimens

Anti-human leukocyte antigen (HLA) antibodies are associated with several complications in solid organ transplantations, but their impact after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not yet well defined. To evaluate the relevance of anti-HLA antibodies, we have retrospectively analyzed 107 peripheral blood allo-HSCTs after reduced-intensity conditioning regimen between 2005 and 2010. Acute myeloid leukemia and multiple myeloma were the most frequent malignancies in the cohort. The detection of anti-HLA antibodies was systematically performed in all patients before transplantation. Anti-HLA antibodies were present in 24 patients (22%). There was no significant impact of anti-HLA antibodies on engraftment, incidence of relapse, and incidence of acute graft-vs-host disease. The presence of anti-HLA antibodies was associated with significantly worse overall survival (p = 0.006) and event-free survival (p = 0.024) after stratification on sex. The 3-year probability of overall survival was 34% without anti-HLA antibodies and 16% in their presence. Patients with anti-HLA antibodies had a higher transplant-related mortality associated with life-threatening vascular complications. Our study supports that anti-HLA antibodies should be tested and considered as an important impacting factor for transplantation outcomes after reduced-intensity conditioning allo-HSCT. We recommend its consideration before allo-HSCT in the donor-recipient selection parameters.

Natalizumab and Impedance of the Homing of CD34&lt;sup&gt;+&lt;/sup&gt; Hematopoietic Progenitors

Treatment with natalizumab, an antibody blocking the α4-integrin, is associated with increased numbers of circulating CD34+ cells in the peripheral blood of patients with multiple sclerosis. To determine whether natalizumab mobilizes CD34+ cells from or inhibits homing to the bone marrow (BM). Fifty-two patients with relapsing-remitting multiple sclerosis treated with natalizumab were included. Flow cytometric analyses; polymerase chain reaction assays for JC (John Cunningham) virus DNA detection; and adhesion, migration, and apoptosis assays of immunomagnetically enriched peripheral blood and BM CD34+ cells were conducted. A comparison was made with CD34+ cells from granulocyte colony-stimulating factor-mobilized peripheral blood or steady-state BM of age- and sex-matched healthy donors. We found adhesion and migration of peripheral blood-derived CD34+ cells to be reduced. In BM aspirates from natalizumab-treated patients, the cellularity, the proportion, and the adhesive capacity of CD34+ cells were normal. The JC virus was undetectable. Natalizumab mediates an increase in circulating CD34+ cells by interfering with homing to the BM. Thus, CD34+ cells appear unlikely to represent a source mobilizing JC virus out of the BM in patients treated with natalizumab.