Clinical Hematopoietic Stem Cell Transplantation Research Articles

Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning

AbstractSuccessful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL–dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.

Hematopoietic stem cell transplantation practice variation among centers in the Eastern Mediterranean Region (EMRO): Eastern Mediterranean Bone Marrow Transplantation (EMBMT) group survey

This practice survey is conducted to analyze clinical hematopoietic stem cell transplantation (HSCT) practice variability among centers in the WHO Eastern Mediterranean Region (EMRO), as represented by the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) group. This internet based survey was completed by the medical program directors of the EMBMT centers; 17 centers participated. The survey collected data on various clinical aspects of HSCT practice. Consistency in pre HSCT cardiac (100%), pulmonary (82%) and viral screen (100%) was observed. Obtaining informed consent was universal. Pre-HSCT psychological assessment is practiced in 50% of the centers. All centers used single-bedded rooms with HEPA filters. Visitor policy during neutropenic phase and the use of gowns, masks or gloves when examining patients varied among centers. MRSA/VRE screen and use of low bacterial diet were applied in 65% and 82%, respectively. Anti-bacterial prophylaxis is employed in 58% (Auto-SCT) and 60% (Allo-SCT) of the centers. Drug choice varied (cotrimoxazole, ciprofloxacin, levofloxacin, piperacillin-tazobactam); 60% of the centers used penicillin prophylaxis in GVHD patients. PCP prophylaxis is applied in 58% (Auto-SCT) and 87% (Allo-SCT) of the centers; cotrimoxazole is usually used. Anti-viral prophylaxis with acyclovir or, less commonly, valacyclovir is used in 70% (Auto-SCT) and 93% (Allo-SCT) of centers. Anti-fungal prophylaxis is applied in 70% (Auto-SCT), 93% (myeloablative Allo-SCT) and 87% (reduced intensity [RIC] Allo-SCT). Fluconazole is used in all Auto-SCT and majority of Allo-SCT recipients; few centers used other agents (itraconazole, voriconazole, amphotericin B) in Allo-SCT. Prophylactic GCSF use varied among centers: Auto-SCT 77%, myeloablative Allo-SCT 33%, RIC Allo-SCT 27%. Use of ursodeoxycholic acid for venoocclusive disease (VOD) prophylaxis is variable: 60% (Allo-SCT) and 12% (Auto-SCT). Cyclosporine/methotrexate is the most commonly used GVHD prophylaxis in myeloablative Allo-SCT (93%); heterogeneity was seen in RIC SCT. Treatment of steroid refractory acute GVHD varied (ATG 53%, higher steroid dose 40%). CMV monitoring varied between antigenemia (53%) and PCR (40%) techniques. Pre-emptive anti CMV therapy is used in 86% of the centers, while 7% used routine CMV prophylaxis; 7% had no specific CMV management policy. Consistency was observed in areas of pre-SCT work up, use of single rooms, HEPA filters and GVHD prophylaxis. Heterogeneity is observed in other practice aspects including other isolation measures, anti-microbial prophylaxis, VOD prophylaxis, growth factor use and treatment of steroid refractory GVHD. Further studies are needed to probe the impact of such practice variations on post-transplant outcome and to ascertain the best clinical practice approach.

New insights into the normal and leukemic stem cell niche: A timely review

New insights into the normal and leukemic stem cell niche: A timely review

Superior Immunomodulatory Effects of Cultured Mast Cells: Potent Strategy for Treatment of Gvhd

AbstractAbstract 4684Mast cells have long been known as effector cells in the various IgE-mediated allergic responses. However, recent studies demonstrated that mast cells play various roles in immune reactions in coordination with other immune cells. That is, mast cells exert pro-inflammatory or anti-inflammatory effects depending on the situation. In addition, mast cells have association with tumor development. In allogeneic hematopoietic stem cell transplantation (HSCT), only a few have reported that the numbers of mast cells were correlated with the severity of acute GVHD in the skin. However, exact role of mast cells in GVHD remains unclear. With the purpose of potential application of mast cells in a clinical HSCT for GVHD, mixed lymphocyte reaction (MLR) was performed to clarify whether mast cells impaired the alloreaction or not. To generate bone marrow derived cultured mast cells (BMCMCs), BM cells from mice were incubated in complete RPMI containing IL-3 for 6 weeks. As shown in the figure, we showed that MLR was strongly inhibited when BMCMCs from the stimulator strain were added to the coculture (Stimulator (S): DCs obtained from C57BL/6, Responder (R): splenocytes from Balb/c, BMCMCs from C57BL/6). Next, when BMCMCs from the responder strain were added to the coculture, MLR was also suppressed (S: DCs obtained from C57BL/6, R: splenocytes from Balb/c, BMCMCs from Balb/c).In conclusion, mast cells suppressed lymphocyte proliferation induced by allo-DCs in an MHC-independent manner. Just like mesenchymal stem cells, cell therapy utilizing cultured mast cells may reduce GVHD severity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Ex vivo expansion of hematopoietic stem cells: mission accomplished?

A small number of hematopoietic stem cells (HSCs) with self-renewal and multi-lineage repopulation capacity maintain hematopoiesis during the lifetime of an individual. Moreover, HSCs and their potential exist in excess as one individual can share its HSCs with another leading to creation of a genetically identical hematopoietic system. For over half a century this property of HSCs has been utilised by successful allogeneic clinical HSC transplantation for treatment of patients with inherited or acquired genetic and neoplastic diseases of the hematopoietic and immune system. There are now more than twenty thousand allogeneic HSC transplants per year worldwide [1]. However, although more than 17.5 million potential HSC donors are registered and additional 500,000 cord bloods are stored for potential allogeneic HSC transplantation [2], timely availability of appropriately human leukocyte antigen (HLA)-compatible HSCs with sufficient quality for patients still poses a problem in the field. Even if a donor is available, toxicity of the procedure could be reduced by increasing HSC numbers in transplants. One way to solve these issues would be by generation of quality-controlled, off the shelf HSC products via in vitro HSC expansion, a "holy grail" procedure many have been hunting for. Here, we discuss accumulating knowledge on signalling pathways involved in HSC maintenance as well as recent achievements to apply the findings to ex vivo HSC expansion for clinical use. Although the specific issue concerns only highly specialised medicine today, newly generated knowledge will be critical for the whole field of stem cell transplantation and regenerative medicine in the future.

Racial differences in presentation, referral and treatment patterns and survival in adult patients with acute myeloid leukemia: A single-institution experience

BackgroundDisease presentation and outcomes differ by race in a number of malignancies, but data in adult acute myeloid leukemia (AML) are limited. Materials and methodsWe conducted a retrospective analysis of pretreatment characteristics, referral and treatment patterns, and outcomes in 548 AML patients evaluated at the University of Maryland Greenebaum Cancer Center, a tertiary care referral center in Baltimore, MD, from 2000 through 2009. Cases were analyzed for time from diagnosis to referral, age, race, gender, socioeconomic status, antecedent hematologic disorder, cytotoxic or radiation therapy for prior malignancy, karyotype, fms-like tyrosine kinase receptor-3 (FLT3) mutations, intensive chemotherapy, clinical trial participation, hematopoietic stem cell transplantation (HSCT) and overall survival (OS). ResultsBlack patients (n=105) were younger than white patients (n=396) (54 vs. 61 years, p<0.001), were more commonly female (55% vs. 45%, p<0.001), and had a lower estimated median household income ($42,677 vs. $53,534 per year, p<0.001). Black patients more frequently had complex karyotypes (26% vs. 12%, p=0.002) and less frequently normal karyotypes (27% vs. 42%, p=0.02). FLT3 mutation frequency was similar. Time to referral and proportion of patients receiving intensive chemotherapy did not differ, but both clinical trial participation (43% vs. 54%, p=0.04) and HSCT (17% vs. 35% for patients ≤70 years old, p=0.001) were less frequent in blacks than whites. Nevertheless, OS was similar in all black and white patients (median 15 vs. 14 months, p=0.23), and when stratified by age, gender and karyotype risk classification. ConclusionAML presentation and treatment differed in black and white patients, but OS was similar. Black patients appear to have barriers to clinical trial participation and HSCT, and there may be barriers to tertiary care referral for black males.

Immunogenetics of hematopoietic stem cell transplantation: the contribution of microsatellite polymorphism studies

Polymorphisms of short tandem repeats of <10 nucleotides, or microsatellites (Msat), are largely used for post-transplant chimerism analyses in clinical hematopoietic stem cell transplantation (HSCT). Compared to single nucleotide polymorphisms (SNP), they have the advantage of a higher degree of allelic polymorphism and thus a potentially larger degree of informativity. Msat markers contribute to approximately 3% of the human genome and have been highly informative in disease association studies, population genetics, forensic medicine and organ and HSC transplantation. They allowed to expand our knowledge of the haplotypic structure of the HLA complex, including the noncoding sequences in the MHC, and to reach a better characterization of immunological phenotypes. Among the different immunogenetic studies in HSCT patients reviewed here, four Msat loci linked to cytokine genes have been analysed by a number of laboratories as potential candidates markers for HSCT outcome: IFNG, TNFd, IL-10(-1064) and IL-1RN. The low patient numbers and high diversity of clinical parameters account for some heterogeneity of the results. Among the trends starting to emerge from these studies, specific TNFd Msat alleles seem to be associated with acute graft-versus-host disease and mortality. Patient/donor Msat incompatibilities have also been used as surrogate markers to map biologically relevant polymorphisms, with a main focus on MHC-resident genetic variation. High throughput SNP typing and next-generation sequencing technologies will allow acquisition of large-scale genomic data and should allow refined analyses of clinically relevant genotypes in the transplantation settting, although the heterogeneity of the study cohorts will remain an issue. The analysis of Msat polymorphisms may still have a place in functional studies on the impact of Msat diversity in the control of immune response gene expression.

Cytokine-Induced Killer Cells Facilitate Immune Reconstitution After Allogeneic BMT In Mice.

AbstractAbstract 3719Cytokine-induced killer (CIK) cells are ex vivo–expanded T lymphocytes expressing both natural killer (NK)– and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft versus host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. With the purpose of potential application of CIK cells in a clinical hematopoietic stem cell transplantation, the problem we have to consider next is whether CIK cells could promote an engraftment and facilitate an immune reconstitution. To this end, lethally irradiated BALB/c mice were injected with minimal number of MHC incompatible C57BL/6 bone marrow (BM) cells alone, in which almost half of mice died because of graft failure, or with CIK concurrently. The mice receiving BM plus CIK cells survived with 93% without GVHD, demonstrating that CIK cells significantly promote an engraftment (P<0.05). In particular, recovery of CD3+ T-cells was significantly faster (p<0.05) in the mice receiving BM plus CIK cells than those receiving BM cells alone. Next we further evaluated whether CIK cells also promote an engraftment in the mice receiving non-myeloablative conditioning using low dose total body irradiation. As expected, CIK cells promoted an engraftment and favored immunoreconstitution, especially from the early time point after BMT. In conclusion, our study clearly demonstrated that CIK cells promote an engraftment and facilitate an immure reconstitution without GVHD. As recent studies demonstrated that sufficient number of CIK cells could be expanded even from washouts of cord blood units bags, infusion of CIK cells would be a potent strategy for preventing a graft-failure in clinical settings, especially after cord blood transplantation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Irradiated Bone Marrow Environment Impairs Hematopoietic Stem Cells by Exposure to Tumor Necrosis Factor a.

AbstractAbstract 3724 Background:Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for various disorders including hematopoietic malignancies. Most HSCT requires cytoreductive conditioning such as total body irradiation (TBI) and/or chemotherapy to ensure engraftment of HSCs by emptying recipients' marrow niches. Our preliminary experiments, however, revealed that TBI could induce local inflammation peaking around days 2–3 within marrow environment. Of note is that conditioning regimens widely used in the current HSCT settings are mostly compatible with HSC exposure to inflammatory storm in post-irradiation bone marrow (BM). Although certain inflammatory cytokines have been shown to affect HSC functions in in vitro, it remains unknown whether in vivo exposure to inflammatory BM environment can alter the characteristics of transplanted HSCs. We therefore sought to investigate what effects irradiated BM environment would have on transplanted donor HSCs using murine systems. Methods and Results:We first tested whether infusion of HSCs at varying timing post TBI would affect transplantation outcomes and HSC functions. To this end, fifty HSCs (CD34-negative/low, cKit+, Sca-1+, lineage marker-negative cells) obtained from Ly5.1-B6 mice were transplanted into lethally irradiated Ly5.2-B6 mice with 1 × 106 competitor Ly5.1/5.2-B6 BM cells at day 0, 3, or 5 post irradiation. No mice survived long-term in the group that received transplants 5 days post irradiation, indicating insufficient HSC engraftment and hematopoietic reconstitution. Although hematopoiesis reconstituted in long term survivors was comparable between another two groups (d0 and d3), we found significant difference in donor HSC ability when tested in a competitive repopulation assay using Ly5.1-KSL cells sorted from primary recipients: Test HSCs obtained from mice transplanted 3 days after irradiation showed poor secondary reconstitution ability, suggesting alteration of pre-engraftment HSC functions depending on transplantation protocols. We then tested whether in vivo exposure of HSCs to irradiated BM environment would have negative effects on HSC functions. Test HSCs (400 cells) from Ly5.1-B6 mice were transplanted into Ly5.2-B6 primary recipients at varying time points (day 0, 1, 2, or 3) after lethal-dose TBI. Approximately 24 h later, BM samples were subjected to a competitive repopulation assay to test secondary reconstitution ability in test HSCs that homed to irradiated BM environment. Consequently, test HSCs that were exposed for ≂f24 h to BM environment at day 2 post TBI showed marked impairment in their long-term reconstitution ability. When “BM-homed” HSCs were enumerated 24 h after infusion, we found modestly impaired HSC homing to BM irradiated 2 days prior to transplantation, compared to BM irradiated on day 0, indicating negative effects on HSCs transplanted during a peak phase of inflammatory storm. Next we examined whether inflammatory cytokines were capable of impairing HSC functions. When tested in liquid culture using purified HSCs, both IFN-g and IL-1 had little effects on colony formation. In contrast, TNF-a inhibited colony formation in a dose-dependent manner. When in vivo HSC exposure to irradiated BM environment was tested using TNF-a KO mice as primary recipients, significant improvement of HSC ability was observed, indicating a major role for this inflammatory cytokine in the HSC-inhibitory effect observed within the irradiated host tissues. We finally sought to test if blocking of TNF-a signaling in HSCs at a peak phase of inflammation could lead to better transplantation outcomes. We utilized the peptide previously shown to block TNFR signaling, and confirmed that pre-incubation of HSCs with this molecule did suppress TNF-a induced reactive oxygen species production in HSCs. Studies are ongoing to test if HSCs transplanted in a peak-phase of TBI-mediated inflammation will benefit from this shielding measure. Conclusion:We demonstrated that inflammatory response in BM environment 2–3 days after irradiation could have a negative effect on donor HSCs regarding both homing efficiency and secondary reconstitution ability. These findings provide important implications for developing the measures that enable HSCs to escape from this inhibitory effect to achieve far more improvement in clinical HSCT outcomes. Disclosures:No relevant conflicts of interest to declare.

Acquisition of G 0 state by CD34-positive cord blood cells after bone marrow transplantation

Hematopoietic stem cells are kept in a quiescent state in the hypoxic area of the bone marrow, which is essential for hematopoietic stem cell maintenance. However, when and how hematopoietic stem cells acquire their hypoxic state and maintain quiescence has not been fully elucidated. The aim of this study was to understand this process in human hematopoietic stem cells after bone marrow transplantation. Human CD34-positive cord blood cells were transplanted into nonobese diabetic/severe combined immunodeficient interleukin-2 receptor γ chain knockout mice. Cell cycle and hypoxia assay analyses were performed, to identify changes in the characteristics of human hematopoietic stem cells following transplantation. Quantitative real-time reverse transcription polymerase chain reaction analysis was used to analyze the transcriptional changes accompanying this transition. Engrafted primitive lineage-negative CD34-positive CD38-negative cells acquired hypoxic state and quiescence in the recipient bone marrow between 4 and 8 weeks, and between 8 and 12 weeks after transplantation, respectively. During 4 and 8 weeks after transplantation, changes in the transcription levels of G₀ regulatory factors, such as CCNC and RBL1, and stem cell regulators, such as Flt3, were also seen, which may be related to the characteristic changes in the cell cycle or oxygenation state. Behavioral changes of hematopoietic stem cells in their cell cycle and oxygenation state during and after bone marrow engraftment may provide insights into hematopoietic stem cell regulation, mediating the improvement of clinical hematopoietic stem cell transplantation protocols and the eradication of leukemia stem cells.

Abstract 1935: Myeloid derived suppressor cells generated in the absence of Muc1 can inhibit allo expansion of T cells while preserving graft versus tumor activity

Abstract Allogeneic hematopoietic stem cell (HSC) transplantation is a promising area of therapy for various hematological malignancies like chronic/acute myeloid leukemia, myelodysplasia and lymphomas. Its effectiveness at inducing the graft versus tumor (GVT) effect is however, compromised by the graft versus host disease (GVHD) frequently observed in recipients post transplant. One of the strategies utilized in minimizing the effect of GVHD while still preserving GVT activity has been to target immunosuppressive cell types like Treg cells or myeloid derived suppressor cells (MDSCs), in an attempt to correct the balance between GVHD and GVT. MDSCs have been shown to be able to inhibit GVHD but not much is known about the ability of MDSCs to preserve GVT. We have previously shown that myeloid progenitors lacking Muc1 from Muc1 knockout (KO) C57BL/6 (H-2b) mice generate more effective MDSCs in vitro and in vivo. In this current study, we study the potential of using MDSCs generated in the absence of Muc1 to alleviate T cell expansion while preserving CTL activity. Our preliminary data shows that MDSCs generated from Muc1 KO C57BL/6 (H-2b) bone marrow are able to inhibit C57BL/6 (H-2b) T cell proliferation in an allogeneic mixed lymphocyte reaction, using Balb/c (H-2d) stimulators, more effectively than wildtype (WT) MDSCs. Interestingly, despite the reduced T cell proliferation in the presence of KO MDSCs, the CTL activity of these C57BL/6 T cells was still preserved, as demonstrated by their ability to lyse Balb/c A-20 leukemia cells. These results inidicate that co transplant of MDSCs generated in the absence of Muc1 can alleviate GVHD across fully mismatched major histocompatibility complex (MHC) barriers while still preserving the GVT ability of the allograft. This would also validate the clinical desirability of down regulating MUC1 in HSCs for more effective generation of MDSCs in a clinical allogeneic HSC transplantation setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1935.

TIMP–1 Plays a Functional Role in CD34. + Hematopoietic Stem and Progenitor Cells.

Abstract 1487Poster Board I-510TIMP–1 protein (Tissue Inhibitor of Metalloproteinases) is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem cell homing and mobilization. We first screened gene array data sets of CD34+ human hematopoietic stem and progenitor cells (HSPCs) and found TIMP–1 mRNA expression. In this study we show that TIMP–1 co-localizes with the tetraspanin CD63 and Beta-1-Integrin. Furthermore, we found a functional interaction of TIMP-1 with its receptor complex on G-CSF mobilized HSPCs.All experiments were carried out using highly enriched CD34+ cells. Co-immunoprecipitation shows that TIMP–1 binds to CD63. Using high resolution Stimulated Emission Depletion (STED) microscopy we could confirm co-localization of TIMP-1 and CD63 as well as Beta-1 Integrin and CD63. To further characterize the role of TIMP-1 in the Beta-1-Integrin signaling, we used an antibody specific to the active form of Beta-1- Integrin. Flow cytometric analysis revealed a significantly higher number of active Beta-1-Integrin in TIMP-1 stimulated cells suggesting TIMP-1 to activate the receptor complex on CD34+ cells. For functional analysis of the receptor complex formation, transwell migration assays were performed showing significantly increased migratory capacities of TIMP–1 treated cells. Additionally, TIMP–1 stimulation leads to a significantly increased adhesion rate of CD34+ cells to the fibronectin-coated dish. To assess a potential role of TIMP–1 in apoptosis, CD34+ HSPCs were co-incubated with thapsigargin and TIMP–1 or DMSO as a control. Subsequent flow cytometric analysis of cleaved Caspase-3 revealed a decrease of apoptotic cells in the TIMP-1 treated samples.In summary, we can show that TIMP-1, CD63 and Beta-1-Integrin form a complex on CD34+ HSPCs. TIMP-1 activates the Beta-1-Integrin signaling in HSPCs and alters the adhesive as well as the migratory behavior of CD34+ HSPCs. Furthermore, TIMP-1 induces an antiapoptotic effect in CD34+ cells. The functional effects of TIMP-1 in HSPCs might be of relevance in clinical hematopoietic stem cell transplantation so that we are currently about to verify these effects in an in vivo model. Disclosures:No relevant conflicts of interest to declare.

Immune Reconstitution: How It Should Work, What's Broken, and Why It Matters

Of the multiple cell lineages undergoing reconstitution in the setting of hematopoietic stem cell transplantation (HSCT), this overview will focus on reconstitution of the T cell arm of the immune system. In clinical HSCT, the autologous setting is free of constraints imposed by reactions to allogeneic antigens, but markers are lacking to distinguish reconstitution from transferred cells versus stem or progenitor cells left residual in the host. In allogeneic transplants, the converse is true – the stem cell origins of the reconstituted populations can be determined, but the biology of such reconstitution is confounded by concomitant allogeneic responses. Because of these constraints, the basic biology of T cell immune reconstitution was first delineated in mouse models in which the availability of congenic strains allowed stem cell origins of reconstituted cell populations to be investigated without the presence of confounding allogenic reactions. Two pathways of T cell regeneration were identified: the thymic-dependent maturation of new T cells from marrow progenitors, and thymic-independent expansion of mature peripheral T cells. 1 These initial studies also determined that no other pathways existed by which substantial peripheral populations of T cells were generated, and also established a means for investigating T cell reconstitution in humans by analyzing the expression of isoforms of the leukocyte common antigen CD45 as markers of naive and memory populations which correlated with the pathway of regeneration. They also demonstrated that homeostatic expansion of mature T cells occurred in lymphopenic hosts. Studies of such expansion have formed central concepts of homeostasis – that T cell levels are maintained by a balance between cytokine-consuming cells and homeostatic cytokines that support the maturation, proliferative expansion and survival of T cells.2 In translating these murine findings to humans, the recovery of CD4+ T cells in pediatric patients who were severely lymphopenic following chemotherapy treatment were initially tracked (Figure 1). There was a strong correlation between the recovery of total CD4+ T cell numbers and the recovery of naive, CD45RA+CD45RO- CD4+ T cells after as few as six months. 3 Furthermore, recovery of naive CD4+ cells was associated with a marked expansion of thymus volume, consistent with a dynamic regulation of the thymus.

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care. Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease. Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO(+) CD27(-)) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25(high) Foxp3(+)) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease. Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.

Graft Versus Host Disease (GVHD) Prophylaxis with Everolimus and Tacrolimus Is Associated with a High Incidence of Sinusoidal Obstruction Syndrome and Microangiopathy – Results of the EVTAC Trial

Beyond disease biology, the success of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematological malignancies is mainly determined by the occurrence and extent of graft versus host disease (GVHD). Therefore, prevention of GVHD is the major goal and challenge in clinical HSCT. A calcineurin-inhibitor combined with methotrexate is the standard graft versus host disease (GVHD) prophylaxis after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus is a derivative of sirolimus, which also seems to mediate anti-leukemia effects. Given the potential synergism and favourable toxicity profile of everolimus and tacrolimus (EVTAC) after allogeneic HSCT we sought to investigate the efficacy of this combination in patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). We report a combination of everolimus (days 0–56) and Tacrolimus (from day −1 on) in 24 patients (pts, median age 62 years) with either MDS (n=17) or AML (n=7) undergoing intensive busulfan-based conditioning followed by HSCT from related (n=4) or unrelated matched (n=12) or 1-allele mismatched (n=8) donors. All pts engrafted and only one experienced grade IV mucositis. However, although everolimus was scheduled to be administered up to day 56, patients received the drug a median of 44 days (range 10–56) only. The reason for premature discontinuation (50%) were either occurrence of early-onset (day 6) GVHD associated hyperbilirubinemia CTC grade 4 (n=1), transplantation-associated microangiopathy (TMA, n=3), sinusoidal obstructive syndrome (SOS) of the liver (n=6) or a drop of platelets after engraftment by at least 50% (n=2). Nine pts (37%) developed grade II–IV acute GVHD, however, chronic extensive GVHD was observed in 11 of 17 (64%) evaluable pts. TMA occurred in 7 pts (29%) with two cases of acute renal failure. In five out of seven patients with TMA either tacrolimus (n=4) or everolimus (n=1) blood through levels were slightly above the upper target level at the time of TMA appearance. The study was terminated prematurely because additional 6 pts (25%) developed SOS, which was fatal in two cases. With a median follow-up of 26 months, the 2-year overall survival rate is 47%. In conclusion, although this new combination appears to be effective as prophylactic regimen for acute GVHD, the incidence of TMA and SOS seems to be higher compared to other regimens. As a result this combination cannot be recommended as prophylactic regimen after busulfan-based intensive conditioning. However, studies in the context of TBI-based or reduced-intensity conditioning regimens might come to a different conclusion.

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.

The Effects of Rapamune on NK Cell Post Bone Marrow Transplantation in Mice.

Natural killer(NK) cells are innate effector cells of the immune system, believed to limit viremia and tumor burden before the onset of adaptive T and B cell immunity. NK cells are potent effector cells in allogeneic bone marrow transplantation. NK cell activity is partially controlled through interactions between killer Ig-like receptors on NK cells and their respective HLA class I ligands. Immunosuppressive agents including cyclosporin, FK506 and Rapamune are utilized in clinical hematopoietic stem cell transplantation. Little is known about the effects of immunosuppressive agents on NK cells post bone marrow transplantation. The in vivo effects of Rapamune on NK cells were determined in an allogeneic bone marrow transplantation model. Splenic NK cell levels in recipients treated with Rapamune decrease markedly. NK cell proliferation and function are significantly decreased in the presence of Rapamune. Studying the differential effects of immunosuppressive drugs on NK cell function is critical in clinical hematopoietic stem cell transplantation.

CD11b-Donor Dendritic Cells Elevate Donor T-Cell Production of Interferon-Gamma and Graft-Versus-Leukemia Activity in Allogeneic Bone Marrow Transplantation.

Host antigen-presenting cells (APC) persist after high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) and initiate graft-versus-host disease (GvHD) in mouse models of HSCT. The role for donor APC on transplant outcomes is less clear. In clinical allogeneic HSCT from HLA-matched siblings, larger numbers of donor plasmacytoid dendritic cell (DC) precursors were associated with more relapse, and worse survival. Depletion of CD11b+ cells from bone marrow (containing CD11b+ DC) modestly augmented graft-versus-leukemia (GvL) activity in murine allogeneic HSCT. In this study, using allogeneic MHC mis-matched HSCT (C57BL/6→B10.BR) of mice bearing a lymphoblastic leukemia (LBRM), recipients of FACS–purified CD11b− donor DC plus FACS–purified HSC and T-cells had dramatically improved long-term survival (45% alive at >100 days) compared to d 5% survival among recipients of HSC and T-cells, or HSC, T-cells and CD11b+ DC (p<0.001). Both donor CD11b− and CD11b+ DC homed to lymphoid organs, and physically contacted donor T-cells, but only the CD11b− DC subset led to higher levels of interferon-γ (IFN- γ) in serum and higher levels of donor spleen-derived T-cells that synthesized IFN-γ in vivo in the first 10 days post-transplant. In contrast, recipients of CD11b+ DC had higher level of sera of IL–10. In CD11b− DC recipients, donor effector-memory CD8+ T-cells proliferated more rapidly and expanded in recipients, but did not cause debilitating GvHD in mice transplanted without leukemia. Donor-spleen-derived T-cells among recipients of CD11b− DC were predominately CD8+ (CD4:CD8 ratio 0.7:1), while donor spleen-derived T-cells were predominately CD4+ (p<0.02; CD4:CD8 ratio 1.3:1) among recipients of CD11b+ DC. In vitro co-culture of purified DC with homologous T-cells responding to allo-antigen demonstrated the same pattern of cytokine production as found in vivo, supporting the ability of CD11b− DC to generate Th1 immune responses that are associated with enhanced GvL effects and improved immune reconstitution. In vitro stimulation of DC subsets by CD40L (1μg/mL) and LPS (1μg/mL) showed that CD11b+ DC had higher level of PD–L1 expression compared to CD11b− DC. In vitro exposure of LBRM to IFN-γ, at doses of 10–300 pg/ml, similar to those observed in vivo, had no direct cytotoxic effect, and had no long-term growth-inhibitory effect on proliferation during 5 days of culture. CD11b+ DC expressed higher levels of PD–L1 and led to higher T-cell synthesis of IL–10 in vivo and in vitro. Transplantation of CD11b− DC resulted in higher levels of donor T-cell production of IFN-γ, and enhanced the GvL effect of donor T-cells without producing debilitating GvHD. These data indicated that purified donor DC can regulate post-transplant immunity via indirect antigen presentation to donor T-cells. CD11b+ DC expressed higher level of PD–L1 and led to higher level of IL–10 that resulted in more relapse and light GvHD. CD11b− DC led to higher level of donor IFN-γ that resulted in a stronger GvL effect while no debilitating GvHD. Engineering the DC content of an allograft may augment immune reconstitution and GvL activity without significantly increasing GvHD in allogeneic HSCT.

How Glycosylphosphatidylinositol-phospholipase D acts in homing of hematopoietic stem/progenitor cells?

Homing of hematopoietic stem/progenitor cells (HS/PC) to the bone marrow (BM) microenvironment is the first and essential step in HS/PC engraftment and initiation of the marrow reconstitution during clinical hematopoietic stem cell transplantation (HSCT). How to improve the homing efficiencies and make full use of HS/PC resources, especially umbilical cord blood (UCB), are of great importance in clinical practice. However, the cellular and molecular mechanisms that govern this process are poorly understood. Glycosylphosphatidylinositol-phospholipase D (GPI-PLD) is an enzyme which can regulate the expression of Glycosylphosphatidylinositol (GPI)-anchored proteins and modulate their correspondent functions by releasing GPI-anchored proteins from cell membrane. Recent studies suggested that the mechanisms of the malignancy and prognosis of certain tumors were correlated with GPI-PLD. HS/PC homing was similar to tumor invasion and metastasis in some process. Here we proposed the hypothesis that GPI-PLD might also has played a role in the homing of HS/PC by modulating the adhesion and migration of these cells. If GPI-PLD did participate in HS/PC homing, maybe the mechanisms of homing can herefrom be partly elucidated, which would benefit transplantation in clinical practice.

Long-term engraftment of p18 INK4C-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18(INK4C) (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18(-/-) HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18(-/-) HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.