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Allogeneic haematopoietic stem cell transplant (HSCT) recipients remain at high risk of adverse outcomes from coronavirus disease 2019 (COVID-19) and emerging variants. The optimal prophylactic vaccine strategy for this cohort is not defined. T cell-mediated immunity is a critical component of graft-versus-tumour effect and in determining vaccine immunogenicity. Using validated anti-spike (S) immunoglobulin G (IgG) and S-specific interferon-gamma enzyme-linked immunospot (IFNγ-ELIspot) assays we analysed response to a two-dose vaccination schedule (either BNT162b2 or ChAdOx1) in 33 HSCT recipients at ≤2 years from transplant, alongside vaccine-matched healthy controls (HCs). After two vaccines, infection-naïve HSCT recipients had a significantly lower rate of seroconversion compared to infection-naïve HCs (25/32 HSCT vs. 39/39 HCs no responders) and had lower S-specific T-cell responses. The HSCT recipients who received BNT162b2 had a higher rate of seroconversion compared to ChAdOx1 (89% vs. 74%) and significantly higher anti-S IgG titres (p = 0.022). S-specific T-cell responses were seen after one vaccine in HCs and HSCT recipients. However, two vaccines enhanced S-specific T-cell responses in HCs but not in the majority of HSCT recipients. These data demonstrate limited immunogenicity of two-dose vaccination strategies in HSCT recipients, bolstering evidence of the need for additional boosters and/or alternative prophylactic measures in this group.

Background Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogeneic stem cell transplantation. These patients face unique challenges due to the complexity of GVHD which can affect multiple organ systems, and the toxicity of treatments. Despite the known impact on quality of life (QOL), qualitative data within the bone marrow transplantation (BMT) literature is rare, and there has been no qualitative work exploring patient experience of specialist healthcare provision for GVHD in the United Kingdom. Methods We conducted a primary explorative qualitative study of the experience of QOL issues and multidisciplinary care in patients with chronic GVHD following allogeneic stem cell transplantation. Eight patients were identified using convenience sampling from specialist BMT outpatient clinics. Following consent, patients were interviewed individually via telephone. Transcripts of interviews were analyzed using an inductive thematic approach. Results Mean participant age was 61-years-old (range 45–68), with a mean time post-transplant of 3 years at time of interview (range 3 months–15 years). Five key QOL themes were identified: (1) ‘Restricted as to what I can do’; (2) Troubling symptoms—‘you can sort of get GVHD anywhere’; (3) Confusion/uncertainty over GVHD symptoms—‘Is this the GVHD?’; (4) Unpredictable course and uncertainty about the future; and (5) Adapting to the sick role. In addition, four themes related to experience of service provision were identified: (1) personal care and close relationship with BMT nurses; (2) efficiency versus long waits—‘On the case straight away’; (3) information provision—‘went into it with a bit of a rosy view’; and (4) the role of support groups. Conclusions These qualitative data reflect the heterogeneity of experiences of the GVHD patient population, reflecting the need for a flexible and nuanced approach to patient care with emphasis on comprehensive information provision. We have identified the key role that BMT specialist nurses within the multidisciplinary team play in supporting patients. We advocate future research should focus on ways to meet the complex needs of this patient group and ensure that the personal care and close relationships are not lost in service redesigns embracing remote consultations.

Background: Allogeneic haematopoietic stem cell transplantation (HCT) is a curative option for haematopoietic diseases. However, a major limitation to the success of HCT is graft-versus-host disease (GvHD). The use of T regulatory cells (Tregs) as prophylaxis or treatment for GvHD has now emerged with a number of active clinical trials. Whilst many studies use Tregs from the original HCT donor, the future of this cell therapy could be third-party Tregs. Cord Blood (CB), with its established banks, represents a potential source of third-party cells. However, isolating a high purity Treg cells from cryopreserved CB is difficult without employing flow sorting. Here we investigated the feasibility of using reversible streptamer technology-based selections to obtain clinical grade Tregs from cryopreserved CB units.Results: A streptamer-based Treg selection was developed with both single-step, CD25 only positive cell selection and two-step, CD4 then CD25 positive selection from cryopreserved CB units, to yield a method that can be readily adapted to full good manufacturing practice (GMP). The best purity was achieved with a two-step streptamer isolation method, giving median purities of 89% Tregs of total cells. This method took advantage of the reversible nature of the streptamer technology allowing for successive positive selections for CD4+ and then CD25+ cells from cryopreserved CB units in an enclosed bag system. Isolated Tregs subsequently demonstrated 300-fold culture expansion using anti-CD3/28 beads. The expanded cells contained high proportions of cells with a Treg phenotype (both by flow cytometry and epigenetics) and demonstrated suppressive function.Conclusions: Using streptamer selection, highly enriched Tregs could be isolated from cryopreserved CB. The method could be performed in an enclosed bag system utilizing readily available clinical processing materials. Moreover, Tregs selected in this manner could be expanded in culture to make a clinically relevant dose from a cryopreserved CB unit. Thus, streptamers represent a viable alternative to column based magnetic bead or fluorescent activated cell sorting (FACS)-based methods for selection of clinical grade Tregs from banked CB units.

Outcomes in acute myeloid leukemia (AML) are dependent on patient- and disease-characteristics, treatment, and socioeconomic factors. AML outcomes between resource-constrained and developed countries have not been compared directly. We analyzed two cohorts: from São Paulo state, Brazil (USP, n = 312) and Oxford, United Kingdom (OUH, n = 158). USP cohort had inferior 5-year overall survival compared with OUH (29% vs. 49%, adjusted-p=.027). USP patients have higher early-mortality (23% vs. 6% p

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1-12 months post-transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti-thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (

BackgroundAllogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders. However, it carries significant risk of morbidity and mortality. To improve patient outcomes, better graft selection strategies are needed, incorporating HLA matching with clinically important graft characteristics. Studies have shown that the cellular content of HCT grafts, specifically higher ratios of T regulatory (Tregs)/T cells, are important factors influencing outcomes when using adult peripheral blood mobilised grafts. So far, no equivalent study exists in umbilical cord blood (CB) transplantation due to the limitations of cryopreserved CB samples.Study design and methodsTo establish the most robust and efficient way to measure the Treg content of previously cryopreserved CB units, we compared the enumeration of Treg and CD3+ cells using flow cytometry and an epigenetic, DNA-based methodology. The two methods were assessed for their agreement, consistency and susceptibility to error when enumerating Treg and CD3+ cell numbers in both fresh and cryopreserved CB samples.ResultsEpigenetic enumeration gave consistent and comparable results in both fresh and frozen CB samples. By contrast, assessment of Tregs and CD3+ cells by flow cytometry was only possible in fresh samples due to significant cell death following cryopreservation and thawing.ConclusionEpigenetic assessment offers significant advantages over flow cytometry for analysing cryopreserved CB; similar cell numbers were observed both in fresh and frozen samples. Furthermore, multiple epigenetic assessments can be performed from DNA extracted from small cryopreserved CB segments; often the only CB sample available for clinical studies.

Allogeneic haematopoietic stem cell transplant (allo-HSCT) offers potentially curative therapy for patients with relapsed/refractory lymphoid malignancies. Reduced-intensity conditioning (RIC) with Alemtuzumab reduces transplant-related mortality and graft-versus-host disease (GvHD), but may be associated with increased risk of relapse. With the aim of studying the effect of GVHD and donor lymphocyte infusions (DLI) on relapse, we performed a retrospective study of 288 patients (57% non-Hodgkin lymphoma, 24% Hodgkin lymphoma and 19% chronic lymphocytic leukaemia; 58% were relapsed/refractory) who underwent RIC-Alemtuzumab-HSCT between 2000 and 2012. Median follow-up time for survivors was 64 months. Five-year overall survival, relapse incidence, GvHD/relapse-free survival and non-relapse mortality were 47%, 33%, 37% and 28% respectively. Cumulative incidence of grade II-IV acute and extensive chronic GvHD was 22% and 21% at 100 days and 5 years respectively. On multivariate analysis, presence of GvHD (P = 0·03) and unrelated donor type (P = 0·03) were protective of relapse. 62/288 patients received DLI for either mixed donor chimerism (prophylactic DLI, n = 37) or clinical relapse (therapeutic DLI, n = 25). Prophylactic and therapeutic DLI successfully converted the patient to full or stable mixed donor chimerism in 78% and 56% of patients respectively. These data demonstrate good long-term outcomes and support the concept of the graft-vs-lymphoma effect as a key protective factor against relapse following RIC-Alemtuzumab allo-HSCT for patients with mature lymphoid malignancies.

Several studies have reported an impact of adult hematopoietic stem cell donor cytomegalovirus (CMV) serostatus on allogeneic hematopoietic cell transplantation outcomes. Limited data, however, are available on the impact of cord blood unit (CBU) CMV serostatus on allogeneic umbilical cord blood transplantation (UCBT) outcomes. We analyzed, retrospectively, the impact of CBU CMV serostatus on relapse incidence (RI) and 2-year nonrelapse mortality (NRM) of single-unit CBU transplantation for acute leukemia. Data from 1177 de novo acute leukemia pediatric and adult patients transplanted within European Group for Blood and Marrow Transplantation centers between 2000 and 2012 were analyzed. CBUs were provided by the European Cord Blood Banks. The median follow-up time for live patients was 59.9 months. The recipients of CMV-seropositive and -seronegative CBUs showed a comparable RI (33% versus 35%, respectively, P = .6) and 2-year cumulative incidence of NRM (31% versus 32%, respectively, P = .5). We conclude that CBU CMV serostatus did not influence RI and NRM in de novo acute leukemia patients after allo-UCBT and should not be included as a criteria for cord blood choice.

Allogeneic haematopoietic stem cell transplant (HSCT) recipients are at increased risk of morbidity and mortality, often due to the development of acute or chronic graft-versus-host disease (GVHD). Low numbers or proportions of regulatory T cells (Tregs) have been reported in patients who develop GVHD. We undertook a systematic review of studies that reported the Treg composition of HSCT grafts in patients with haematological malignancies. Fourteen eligible studies were identified, eight of which stratified patients by Tregs (absolute dose or ratio to CD3+ or CD4+ cells). Meta-analyses showed that high levels of Tregs in the grafts were associated with improved overall survival [hazard ratio (HR) 0·42, 95% confidence interval (CI) 0·23-0·74, P = 0·003, 2 studies], with a significant reduction in non-relapse mortality (HR 0·30, 95% CI 0·14-0·64, P = 0·002, 2 studies) and a reduced risk of acute GVHD (relative risk (RR) 0·59, 95% CI 0·40-0·89, P = 0·01, 6 studies). The consistency of these findings strongly suggests that the Treg composition of HSCT grafts has a powerful effect on the success of allogeneic HSCT. The major challenge is to translate these findings into better selection of allografts and future donors to provide a substantial improvement in allogeneic HSCT outcomes and practice.

Background Allogeneic haematopoietic cell transplantation (HCT) is a curative therapy for severe haematological disorders, including acute leukaemia. However, it carries significant morbidity and mortality due to infection, graft-versus-host disease and relapse. The cellular content of HCT grafts, in particular the ratio between regulatory T cells (Treg) and conventional T cells, is an important factor influencing the severity of these complications. Whilst previous studies have been performed in fresh adult peripheral blood mobilised grafts, it has not been applied in umbilical cord blood (CB) HCTs. CB enumerations are more challenging as they must be performed from small cryopreserved segments stored alongside the CB units used for HCT. Study and design methods Fresh CB units and thawed segments were analysed for their Treg and T cell content using both flow cytometry (the benchmark technique) and an epigenetic, DNA-based methodology. The two methods were compared for their agreement, consistency and susceptibility to error when assessing Treg and CD3+ cell numbers in both fresh and cryopreserved samples. Results Epigenetic enumeration gave consistent results in both fresh and frozen samples, providing Treg/CD3 estimates that were similar. Assessment of Tregs and CD3+ cells by flow cytometry and epigenetic assessments in fresh samples showed that these two methods were correlated. Conversely, significant cell death was observed in the thawed segments which affected Treg and CD3 cell estimates by flow cytometry. Conclusion Epigenetic assessments offer significant advantages over flow cytometry for analysing cryopreserved CB. Unlike with flow cytometry assessments of thawed segments, similar cell numbers were observed in fresh and frozen samples, with material requirements not being limiting and being unaffected by high cell death. With this method, multiple epigenetic assessments can be performed from extracted DNA, to provide statistical confidence and confirm observed results. Finally, the method raises the possibility of retrospective studies of historical samples where only DNA is available.

Malignancy is a micro-evolutionary phenomenon shaped by selection pressures. Chief among these is the adaptive immune system, which recognizes malignant cells as a threat and attempts to eradicate them. The task is not easily achieved – if it were, cancer would not be a part of our human experience. The field of immunotherapy has rapidly expanded over the last two decades. It has produced some of the most exciting results of 21st century medicine, and has deepened clinicians' understanding of the relationship between malignancy and the immune response. This review discusses this relationship and analyses key tools in the immunotherapy arsenal.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post–allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre–allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post–allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis.

Haematopoietic stem cell transplantation was proposed as a treatment strategy just over 60 years ago. Owing to great advances in the field, haematopoietic stem cell transplantation has become an established method for the treatment of many haemato-oncological, immunological and hereditary conditions with the potential of cure. The number of haematopoietic stem cell transplants performed worldwide reached one million by 2012. This review provides an overview of autologous and allogeneic haematopoietic stem cell transplantation including disease indications, the individual steps of the procedure and outcome, and highlights achievements in the treatment of autoimmune diseases. Although autoimmune conditions account for only 1% of indications for autologous haematopoietic stem cell transplant, this is increasingly used to treat high-risk autoimmune diseases. Haematopoietic stem cell transplantation can induce long-term remission by resetting the immune system via eradication of autoreactive immune cells and the generation of a de novo self-tolerant immune system. Data seem most encouraging in multiple sclerosis and systemic sclerosis and it is likely that the number of procedures performed to treat these conditions will rise in the future.

Summary Background Due to paucity of evidence to guide management of allogeneic haematopoietic stem cell transplantation (allo-HSCT) patients with respiratory syncytial virus (RSV) infections national and international guidelines make disparate recommendations. Methods The outcomes of allo-HSCT recipients with RSV infection between 2015 and 2017 were assessed using the following treatment stratification; upper respiratory tract infections (URTI) being actively monitored and lower respiratory tract infections (LRTI) treated with short courses of oral ribavirin combined with intravenous immunoglobulin (IVIG, 2 g/kg). Results During the study period 49 RSV episodes were diagnosed (47% URTI and 53% LRTI). All patients with URTI recovered without pharmacological intervention. Progression from URTI to LRTI occurred in 15%. Treatment with oral ribavirin given until significant symptomatic improvement (median 7 days [3–12]) and IVIG for LRTI was generally well tolerated. RSV-attributable mortality was low (2%). Conclusions In this cohort study, we demonstrate that active monitoring of allo-HSCT patients with RSV in the absence of LRTI was only associated with progression to LRTI in 15% of our patients and therefore appears to be a safe approach. Short course oral ribavirin in combination with IVIG was effective and well-tolerated for LRTI making it a practical alternative to aerosolised ribavirin. This approach was beneficial in reducing hospitalisation, saving nursing times and by using oral as opposed to nebulised ribavirin.

Regulatory T cells (Tregs) are CD4+ T cells that are key players of immune tolerance. They are powerful suppressor cells, able to impact the function of numerous immune cells, including key effectors of inflammation such as effector T cells. For this reason, Tregs are an ideal candidate for the development of cell therapy approaches to modulate immune responses. Regulatory T cell (Treg) therapy has shown promising results so far, providing key knowledge on the conditions in which these cells can provide protection and demonstrating that they could be an alternative to current pharmacological immunosuppressive therapies. However, a more comprehensive understanding of their characteristics, isolation, activation, and expansion is needed to be able design cost effective therapies. Here we review the practicalities of making Tregs a viable cell therapy, in particular, discussing the challenges faced in isolating and manufacturing Tregs and defining what are the most appropriate applications for this new therapy.

Allogeneic haemopoietic stem cell transplantation offers a potentially curative treatment option for a wide range of life-threatening malignant and non-malignant disorders of the bone marrow and immune system in patients of all ages. With rapidly emerging advances in the use of alternative donors, such as mismatched unrelated, cord blood and haploidentical donors, it is now possible to find a potential donor for almost all patients in whom an allograft is indicated. Therefore, for any specific patient, the transplant physician may be faced with a myriad of potential choices, including decisions concerning which donor to prioritize where there is more than one, the optimal selection of specific umbilical cord blood units and which conditioning and graft-versus-host disease prophylactic schedule to use. Donor choice may be further complicated by other important factors, such as urgency of transplant, the presence of alloantibodies, the disease status (homozygosity or heterozygosity) of sibling donors affected by inherited disorders and the cytomegalovirus serostatus of patient and donor. We report UK consensus guidelines on the selection of umbilical cord blood units, the hierarchy of donor selection and the preferred conditioning regimens for umbilical cord blood transplantation, with a summary of rationale supporting these recommendations.

Donor lymphocyte infusion, a rescue therapy after hematopoietic stem cell transplantation, has been increasingly adopted, as modalities of stem cell transplantation have widened. First described as donor lymphocyte transfusion or cell therapy, it consists of infusion of donor lymphocytes, collected in steady state or after growth factor enhancement. As in literature the most used name is donor lymphocyte infusion, we'll adopt it here. Its most striking efficacy is observed in patients with chronic myelogenous leukemia, who relapsed after allogeneic stem cells transplantation. However, graft-versus-host disease, its main complication, may still hamper its feasibility.

SUMMARY Objectives To establish rates of cytomegalovirus (CMV) transmission with use of CMV-unselected (CMV-U), leukocyte-reduced blood components transfused to CMV-seronegative patient/CMV-seronegative donor (CMV neg/neg) allogeneic stem cell transplantation (SCT) recipients including those receiving T-depleted grafts. Background CMV infection remains a major cause of morbidity following SCT. CMV-seronegative SCT recipients are particularly at risk of transfusion transmitted CMV (TT-CMV) and until recently they have received blood components from CMV-seronegative donors with significant resource implications. Although leukocyte reduction of blood components is reported to minimise risk of TT-CMV, its efficacy in high-risk situations, such as in T-depleted transplant recipients, is unknown. Methods We retrospectively analysed the incidence of TT-CMV in CMV neg/neg allogeneic SCT recipients transfused with CMV-U, leukocyte-reduced blood components in two transplantation centres in the UK. Patients were monitored for CMV infection by weekly CMV polymerase chain reaction testing. Leukocyte reduction of blood components was in accordance with current UK standards. Results Among 76 patients, including 59 receiving in vivo T-depletion, no episodes of CMV infection were detected. Patients were transfused with 1442 CMV-unselected, leukocyte-reduced components, equating to 1862 donor exposures. Conclusions Our findings confirm the safety of leukocyte reduction as a strategy in preventing TT-CMV in high-risk allogeneic SCT recipients.

SUMMARY Objectives To establish whether passive transfer of cytomegalovirus (CMV) IgG via transfusion results in ambiguous serostatus in patients undergoing allogeneic stem cell transplant (SCT). Background CMV infection causes significant morbidity following allogeneic SCT. Leucocyte-reduced blood products from CMV-seropositive donors carry minimal risk of CMV transmission, however, may result in passive transfer of CMV IgG, leading to unintentionally CMV-mismatched recipient/donors with significant consequences. Methods We undertook a retrospective single-centre analysis of CMV IgG results in patients transfused with CMV-unselected (CMV-U) leucocyte-reduced components subsequently undergoing SCT. Results Of patients with >1CMV IgG measured, 8/29 (27·6%) had discordant results; all were transfused between negative and subsequent positive results and were thought to have passively acquired CMV IgG. One likely CMV naive patient was recorded as CMV seropositive and underwent transplant with a seropositive donor, developing CMV infection which required treatment. Conclusions Passive transfer of CMV IgG is an unanticipated consequence of transfusion of CMV-U products and has the potential to cause morbidity. Inaccurate recording of serostatus pre-transplant has wider implications for data reporting on transplant outcomes. When transfusing CMV-U components pre-transfusion CMV IgG samples must be taken and transfusion history must be considered when interpreting results.

The effect of killer cell immunoglobulin-like receptor (KIR)-ligand matching on outcomes after unrelated cord blood (CB) transplantation was studied in 461 patients with acute myeloid leukemia, categorizing KIR ligand for HLA-C groups C1 and C2 and Bw4. Donor-recipient HLA matching considered allele-level matching at HLA-A, -B, -C, and -DRB1. Separate analyses were conducted for 6-7/8 HLA-matched and 3-5/8 HLA-matched transplants because HLA matching confounded KIR-ligand matching (ie, KIR-ligand mismatching was less likely with better HLA matching). All patients received single CB unit and myeloablative conditioning. There were no significant differences in nonrelapse mortality (NRM), relapse, and overall mortality by KIR-ligand match status. However, among recipients of 3-5/8 HLA-matched transplants, NRM (HR, 2.26; P = .008) and overall mortality (HR, 1.78; P = .008) but not relapse were higher with KIR-ligand mismatched (host-versus-graft direction) compared with KIR-ligand matched transplants. These data do not support selecting CB units based on KIR-ligand match status for transplants mismatched at 1 or 2 HLA loci. Although transplants mismatched at 3 or more HLA loci are not recommended, avoiding KIR-ligand mismatching in this setting lowers mortality risks.

Following allogeneic stem cell transplantation, graft versus host disease (GVHD) is a major cause of morbidity and mortality with the commonest organs affected being the skin and oral mucosa. Clinical presentation of cutaneous GVHD is widely variable and 13/30 patients attending a dedicated GVHD clinic were referred to Dermatologists with a specialist interest in GVHD (1). The British Committee for Standards in Haematology GVHD guidelines recommend organ-specific management and supportive care (2) recognising that early input from a Dermatologist is likely to improve clinical outcomes (3). JACIE 6th Edition standards also recommend access to certified specialist trained Dermatologists (4). This article is protected by copyright. All rights reserved.

Delayed engraftment following cord blood (CB) transplantation remains a significant challenge. While cell dose is clearly limiting, CB derived hematopoietic stem cells (HSCs) also exhibit a deficit in homing and engraftment. IL-15 activated natural killer (NK) cells have been shown as potential promoters of homing and engraftment of CB HSCs. However, the role of NK cells and their underlying mechanisms in promoting CB HSCs requires further study. Here, we explore the effect of IL-15 activated CB NK cells on the functional properties of CB CD34+CD133+CD45lo HSCs. In addition, we define the mechanistic interaction between NK cells and HSCs that may increase CB-HSC engraftment and improve patient outcome post-HSC transplantation. We first determined whether IL-15 activated NK cells could improve HSC function in vitro. Purified CD56+CD3- NK cells from CB were stimulated overnight with IL-15 and cultured at a 1:5 cell ratio with autologous purified CD34+CD133+CD45lo cells (CB HSCs). IL-15 activated NK cells increased CXCR4 expression on CB HSCs when compared to cultures with resting NK cells or CB HSCS alone. As HSCs must migrate to the bone marrow in order to engraft and facilitate long-term immune reconstitution, we next assessed whether NK cells also impact on HSC migration, clonogenicity and proliferation. We found that elevated levels of CXCR4 on CB HSCs cultured with activated NK cells also translated into enhanced chemotaxis towards SDF-1α in vitro. IL-15 activated CB NK cells also increased CB HSC clonogenicity as evaluated by short-term in vitro cultures. The effect of activated NK cells on the clonogenic capacity of CB HSCs was cell dose dependent with the highest effect observed at a ratio of 1:10. To study CB HSC proliferation, CFSE stained CD34+CD133+CD45lo cells were cultured either alone, with resting NK cells or IL-15 activated NK cells. Cultures with IL-15 activated NK cells significantly increased CB HSC proliferation when compared to cultures with resting NK cells or CB HSCs alone [median percentage of proliferating CB-HSCs; 38.4% (34%-44.6%) vs. 46.7% (36%-53.4%) vs. 69% (59.6%-78.5%)]. Moreover, following the ability of IL-15 activated NK cells to upregulate CB HSC proliferation, we investigated whether CB HSCs still retained their long-term engraftment potential. We found that proliferating CB HSCs still recalled both their short-term and long term clonogenic capacity as evaluated by CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. Finally, we demonstrated that IL-15 activated NK cells also possessed the ability to activate pAkt/pErk and pStat3. As pAkt/pErk and pStat3 are key mediators of cell survival proliferation, our findings identify that NK cells may promote the survival and proliferation of CB HSCs through activating pAkt/pErk and pStat3. These data suggest that IL-15 activated NK cells from CB are endowed with properties to promote the functional profile of CB HSCs that contribute to improved engraftment. To further understand the underlying mechanisms through which IL-15 activated NK cells exert their ability to upregulate the functional profile of CB HSCs, we used antibody blockade experiments. We showed that the ability of NK cells to increase CXCR4 expression on CB HSCs was mediated via the provision of IFN-γ, but not TNF-α or TNF-β. Whereas, the effect of NK cells on CB HSC function studied through clonogenicity, proliferation and signalling studies was only partially dependent on IFN-γ production by IL-15 activated NK cells. Using transwell experiments, we further determined that the ability of activated NK cells to upregulate CB HSC function is also partly dependent on direct NK cell/HSC cell contact. Subsequently, we found that the addition of blocking antibody against 2B4 in cultures containing IL-15 activated NK cells and CB HSC partially reversed the ability of NK cells to increase the clonogenic capacity, proliferation and Akt/Erk and Stat3 signalling of CB HSCs. Thus, the ability of IL-15 activated NK cells to increase the functional profile of CB HSCs depends on IFN-γ production and cell-cell contact involving 2B4. Our combined studies demonstrate a novel effect of IL-15 activated CB NK cells and their key factors as potential mediators of stem cell homing and engraftment, which could be utilized to develop strategies that will benefit all patients with haematological malignancies and improve CB transplantation. Disclosures No relevant conflicts of interest to declare.

Umbilical cord blood (CB) is an alternative source of hematopoietic stem cells (HSCs) for patients requiring allogeneic HSC transplantation but lacking a suitable human leukocyte antigen (HLA)-matched donor. Using CB has many advantages, including lower HLA-matching requirements, increased donor availability, and low rates of Graft-versus-Host Disease. Furthermore, with over 600,000 cryopreserved volunteer CB units currently stored in international CB banks worldwide, CB is rapidly available for those patients requiring urgent transplantation. However, concern remains over the low HSC doses available in CB grafts, resulting in delayed engraftment and poor immune reconstitution. This chapter will review the current use and future developments of related and unrelated allogeneic CB transplantation. We will provide an overview of the encouraging results of CB transplantation for the treatment of malignant and nonmalignant conditions, both in adults and children. We will discuss the important factors that need to be considered when selecting CB units for transplantation and introduce some of the new developments that are being currently investigated to further improve the results of CB transplantation.

Background aims There is real and sustained interest in preparing T-regulatory cells from leukapheresis collections for cellular therapy through the use of simple, effective and reliable methods conforming to Good Manufacturing Practice (GMP). We describe a GMP-ready isolation procedure for CD25 + products with the use of a sterile docking device, pigtail sampling, a laminar flow hood and the CliniMACS system that uses leukapheresis collections made by two apheresis machines. Methods We used CD8/CD19 depletion followed by CD25-positive selection. The median number of CD4 + cells recovered was 72.5 ± 32.6 × 10 6 , of which 60.5% ± 17.8% were CD25 + FOXP3 + cells. Suppression of autologous CD25 − cell proliferation by the cryopreserved CD25 + products was 51.3% ± 4.4%, 49.0% ± 3.7% and 39.0% ± 3.6% at CD25 + :CD25 − ratios of 1:1, 1:2 and 1:4 ( n = 6), respectively, comparable to suppression by fresh CD25 + products (53% ± 6.2%, 51% ± 3.3% and 39% ± 7.1%). Results We found Leukapheresis collections by Cobe Spectra contained more lymphocytes and platelets than collections by Spectra Optia apheresis machine (median, 9.2 × 10 9 versus 6.7 × 10 9 ; P = 0.04) and platelets (median, 610 × 10 9 versus 170 × 10 9 ; P = 0.04). The frequency of CD4 + CD25 + FOXP3 + was significantly higher in the leukapheresis (4.85%; 95% confidence interval, 1.95% to 5.38%) than in peripheral blood (3.9%; 95% confidence interval, 2.63% to 6.45%) ( P = 0.02). The CD8- and CD19-negative depletion step was associated with significant loss of total CD4 + T cells ( P = 0.001). Conclusions Results suggest that functional CD25 + products can be isolated with a GMP-ready method, and good recovery can be obtained with the use of an optimized cryopreservation protocol. These data and methods show the potential, possibilities and future work needed to isolate target cell populations in a reproducible, time-efficient and cost-efficient manner for clinical applications.

Umbilical cord blood (UCB) is an important source of haematopoietic stem cells (HSC) for allogeneic transplantation when HLA-matched sibling and unrelated donors (MUD) are unavailable. Although the overall survival rates of UCB transplantation are comparable to the results with MUD, UCB transplants are associated with slow engraftment, delayed immune reconstitution, and increased opportunistic infections. While this may be a consequence of the lower cell dose in UCB grafts, it also reflects the relative immaturity of cellular immunity within cord blood. Furthermore, the limited number of cells and the non-availability of donor lymphocyte infusions (DLI) currently prevent the use of post-transplant cellular immunotherapy to boost donor-derived immunity to treat infection, mixed chimerism and disease relapse. Therefore, to further develop UCB transplantation, many strategies to enhance engraftment and immune reconstitution are currently under investigation. This review summarises our current understanding of engraftment and immune recovery following UCB transplantation and why this differs from allogeneic transplants using other sources of HSC. It also provides an comprehensive overview of the promising techniques being used to improve myeloid and lymphoid recovery, including expansion, homing, and delivery of UCB HSC; combined use of UCB with third party donors; isolation and expansion of NK cells, pathogen specific T cells, and regulatory T cells; methods to protect and/or improve thymopoiesis. As many of these strategies are now in clinical trials, it is anticipated that UCB transplantation will continue to advance, further expanding our understanding of UCB biology and HSC transplantation.

Umbilical Cord Blood (UCB) remains an important alternative source of Hematopoietic Stem Cells (HSC) for allogeneic transplantation when suitable HLA-matched donors are unavailable. Cord Blood (CB) offers many benefits including rapid availability, absence of risk to the donor, and a low incidence of graft-versus-host disease. However, although the overall survival of patients receiving unrelated CB transplants is comparable to using other HSC sources, UCB transplantation is associated with delayed engraftment and poor immune reconstitution, particularly in adults. While this is partly due to the lower cell dose in UCB grafts, it may also reflect the relative immaturity of cord blood. Therefore, many different strategies to enhance hematopoietic engraftment following UCB transplantation are currently under investigation. This article will review the latest techniques including improved collection, HLAmatching, homing and expansion of CB, and the use of double CB grafts, third-party donors, and accessory cells. As many of these methods are now in clinical trials, it is anticipated that UCB transplantation will continue to improve, further expanding our understanding of CB biology and HSC transplantation.

Allogeneic hematopoetic stem cell transplantation (allo-HSCT) offers long-term remission for patients with lymphoid malignancies. In particular, reduced intensity conditioned (RIC) protocols with Alemtuzumab T-cell depletion lead to durable engraftment and reduced graft versus host disease (GvHD). However, such regimens may be associated with reduced graft-versus-lymphoma (GvL) effect and higher incidence of disease progression, leading to increased use of donor lymphocyte infusion (DLI). This study examined transplant outcomes and use of DLI in a large UK RIC-Alemtuzumab conditioned HSCT cohort. 288 consecutive adult patients from three UK transplant centres (Birmingham, Glasgow, Oxford) undergoing first Alemtuzumab-based RIC HSCT for Hodgkin's lymphoma (57%), Non-Hodgkin's lymphoma (24%) and chronic lymphocytic leukemia (19%) between 2000 and 2012 were included. Overall survival (OS), current progression-free survival (CPFS), relapse incidence (RI), transplant-related mortality (TRM), incidence of GvHD and effects of DLI were analyzed. Median age was 49y (range 17-68) and 60% were male. Patients were heavily pre-treated with 58% having received ≥3 prior lines of treatment and 49% having failed a previous autograft. 55% received stem cells from an HLA-matched unrelated donor and 94% received a peripheral blood stem cell harvest. The most frequent conditioning regimen was Fludarabine, Melphalan and Alemtuzumab (FMC) (70%) and the median total Alemtuzumab dose was 50mg (range 30-100). Median duration of follow-up for survivors was 64 months. 3y OS and CPFS were 53% and 50%, respectively. Factors negatively associated with OS in univariate (UV) analysis were previous autograft (p=0.01), no requirement for DLI (p=0.05), disease status not CR (p=0.05), previous treatment number ≥3 (p=0.007) and failure to engraft plts (p=0.001). On multivariate (MV) analysis, only previous autograft (HR 1.58 p=0.01) and failure to engraft plts (HR 2.3 p=0.0001) retained significance. Factors negatively associated with CPFS on UV analysis were no requirement for DLI (p=0.006), failure to engraft plts (p=0.0003) and previous treatment lines ≥3 (p=0.04). On MV analysis, no DLI remained a significant predictor of adverse CPFS (HR 1.8 p=0.01), as did failure to engraft plts (HR 1.77 p=0.005) and ≥3 treatment lines (HR 1.46 p=0.04). At 3y, the cumulative incidence of relapse and TRM was both 27%. The incidence of grade 2-4 acute GvHD (aGvHD) and extensive chronic GvHD (cGvHD) was 22% and 18%, respectively. UV predictors of RI were unrelated donor (p=0.0009), HLA mismatch (p=0.04), age > median (p=0.04) and lack of aGvHD (p=0.04). The presence of cGvHD may have been protective against relapse (p=0.08). No variables retained significance on MV modelling. UV predictors of TRM were failure to engraft plts (p=0.001), HLA mismatch (p=0.005), age > median (p=0.004), previous autograft (p=0.02), FMC conditioning (p=0.03), ≥3 previous lines of treatment (p=0.001) and development of aGvHD (p=0.04). On MV modelling, failure to engraft plt (p=0.001), HLA mismatch (p=0.03), age > median (p=0.003) and treatment number ≥3 (p=0.04) retained significance. 62/288 patients received DLI; 37 receiving prophylactic DLI (pDLI) for mixed chimerism and 25 receiving therapeutic DLI (tDLI) for disease relapse. The median dose of DLI was 1x 10^6 CD3+ cells/kg (range 0.1 - 10 x 10^6). Median time from HSCT to first DLI was 10.5 months (range 3-30), to 2nd DLI 15 months (6-51) and to 3rd DLI 19 months (9-30). At 3y, OS after pDLI was 75% and after tDLI was 29%. 29/37 (78%) of pDLI and 14/25 (56%) of tDLI recipients achieved full or stable mixed chimerism. 3y RI after pDLI was 29% and after tDLI was 59% (p=0.02). Overall, the incidence of GvHD post-DLI was 32% (48% tDLI, 22% pDLI, p=ns). Median time to GvHD after last DLI was 46 days (range 17-249). There was no significant impact of post-DLI GvHD on RI (RI at 1y post DLI 18% in GvHD cohort vs 21% with no GvHD, p=ns), but GvHD increased TRM at 1y post DLI (17% in the GvHD cohort vs 3% with no GvHD, p=0.05). This is the largest series to date with long-term follow-up examining outcomes after Alemtuzumab-based RIC HSCT for mature lymphoid malignancy. Our data indicate that allo-HSCT is effective and is associated with good long term outcomes. Use of pDLI for was associated with excellent outcomes and supports use of DLI in patients developing mixed chimerism following allo-HSCT for lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

We investigated the contributions of methotrexate (MTX) and ciclosporin (CsA) prophylaxis to acute/chronic graft-versus-host disease (a/cGvHD) prevention following reduced-intensity conditioned allogeneic haematopoietic stem cell transplant (HSCT). Ninety-two fludarabine-melphalan sibling allo-SCT received CsA. Nine, 30 and 47 patients received no MTX, 2-3 doses and 4 doses, respectively. Cumulative CsA blood level to day 21 (CsA(21)) was calculated. Grades II-IV aGvHD incidence was 37.2%. In multivariate analysis, MTX omission and increasing donor age significantly associated with aGvHD incidence whilst MTX reduction and CsA(21) did not. Median duration of first immunosuppressive therapy (IST) for aGvHD was 68 days; duration of first IST was significantly longer in older patients but was not associated with MTX or CsA(21). Extensive cGvHD incidence was 60.6% at 1 year. Reduction of MTX to 2-3 doses, but not MTX omission or CsA(21), was associated with greater incidence of cGvHD affecting ≥3 organs. Median IST duration was 22 months; neither MTX nor CsA(21) influenced this. IST duration was significantly greater in patients receiving a CD34 dose below median. Neither MTX nor CsA(21) altered survival or relapse outcomes. MTX influences GvHD following T-replete RIC sibling HSCT.

Co-infusion of donor regulatory T-cells (Tregs) with conventional T-cells (Tcons) in allogeneic haematopoietic stem cell transplantation (HSCT) may improve immune reconstitution and suppress graft-versus-host disease (GvHD) without inhibiting the graft-versus-tumour (GvT) response. While phase I/II studies have focused on co-infusion of ex-vivo isolated Tregs, few have examined the impact of Tregs within peripheral blood stem cell (PBSC) grafts. In previous work, we observed that high proportions of Tregs (CD4+CD25+FOXP3+CD127-/dim T-cells; expressed as percentage of CD4+ T-cells) in PBSC grafts were associated with improved myeloid and platelet recovery and decreased non-relapse mortality (NRM). In this current study, we therefore hypothesised that patients transplanted with high proportions of Tregs within allogeneic PBSC grafts will also have improved lymphocyte recovery. Ninety-four allogeneic HSCT transplants were recruited from Oxford University Hospitals NHS Trust. The median age of the recipients was 48 years (range, 18-74). Forty-seven (50%) of the transplants were performed for acute leukaemia and 80 (85%) were performed using a reduced intensity conditioning regimen. Thirty-eight (40%) PBSC grafts were obtained from sibling donors (37 HLA-matched; 1 single HLA-mismatch (9/10)) and 56 (60%) from unrelated donors (39 HLA-matched (10/10); 17 single HLA-mismatch (9/10)). The cellular contents of the grafts were analysed using multi-colour flow cytometry. The median total nucleated cell (TNC), CD34+, CD3+, CD19+ and CD3-CD56+ cell dose per kg recipient body weight was 10.1 x 108/kg (range, 2.7-37.6), 6.3 x 106/kg (range, 1.1-19.8), 280 x106/kg (range, 87-801), 62 x 106/kg (range, 13-245) and 35 x106/kg (range, 8-101) respectively. Tregs accounted for a median of 2.96% of CD4+ T-cells (range, 0.81-8.56%) within the grafts with a median Treg dose of 4.7 x 106/kg (range, 0.8-20.6). There were no significant correlations between the proportion of Tregs in the grafts and the TNC, CD34+, CD3+, CD19+ or CD3-CD56+ cell dose. The cumulative incidence of total lymphocyte (>1.0 x 109/l), CD3+ (>0.7 x 109/l), CD19+ (>0.1 x 109/l) and CD3-CD56+ (>0.1 x 109/l) cell recovery by 6 months was 71.3% (95% CI, 60.8-79.4%), 51.2% (95% CI, 40.6-62.3%), 62.4% (95% CI, 50.8-72.0%) and 91.0% (95% CI, 82.0-95.7%) respectively. The median time to total lymphocyte, CD3+, CD19+ and CD3-CD56+ recovery was 47 days (range, 11-455), 40 days (range, 33-265), 62 days (range, 27-262) and 34 days (range, 27-173) respectively. Patients transplanted with PBSC grafts containing higher proportions of Tregs (>2.96% CD4+ T-cells (median)) had improved lymphocyte recovery (P These data support the hypothesis that higher proportions of donor Tregs in PBSC grafts may also enhance lymphoid reconstitution following allogeneic HSCT. However, larger studies will be required to understand the potential mechanisms involved and to confirm our findings. Disclosures No relevant conflicts of interest to declare.

In the UK, unrelated cord blood transplantation (UCBT) has been used increasingly in adults since 2000. National guidelines were published in 2009 (Shaw et al, 2009) and two national prospective clinical trials have been established (EUDRACT registrations; RIC 2004-003845-41 and MAC 2009-011818-21). However, national trends and outcomes have never been comprehensively appraised. We have therefore analysed the demographic data and outcomes in adults (>18 years) undergoing UCBT in 23 UK transplant centres from 2000-2012 using the BSBMT and Eurocord databases. From the first adult UCBT in 2000 to the end of 2102 there were a total of 176 centre UCBT registrations with corresponding cord blood bank data, including 28 patients in the national prospective clinical trials which have been excluded from any further analysis. Outcomes were analysed for 148 patients with a median age of 40.8 (range 18-72) years, with acute leukaemia (n=80), myeloproliferative disorders/myelodysplastic syndrome (MDS) (n=43), lymphoproliferative diseases (n=20) or bone marrow failure (n=5). Half the activity was between 2000-2008 and half between 2009-2012, reflecting a greater than doubling of activity in recent years. Various conditioning regimens were used, with the majority receiving a reduced intensity conditioning regimen. Most patients (72%) received double cord blood units (dCBU) and the remainder a single CBU. Recorded median total cell dose infused was 3.61 x107/kg (range 0.41-34.35) for total nucleated cell count (TNC) and 1.69 x105/kg (range 0.13-14.97) for CD34+ cells. Engraftment of neutrophils to >0.5 x109/L occurred at a median of 22 (range 3-52) days and platelets to >20 x109/L at a median of 39 (range 10-117) days. Overall survival at 1 year was 46.4% (CI 38.8-55.5%) and 2 years was 40% (CI 32-49%), with an overall median survival of 27.1 (range 3.2-83.7) months. The incidence of grade II to IV acute graft-versus-host disease (GVHD) was 17.6% (CI 11.5-24.8%) and chronic GVHD at 1 year was 11.4% (CI 6.7-17.6%). In patients treated for malignant disease with remission status available (n=137), cumulative incidence of relapse was 11.0% (CI 6.4-16.9%) at 100 days and 24.6% (CI 17.5-32.4%) at 1 year, and treatment related mortality was 22.6% (CI 16.0-30.0%) at 100 days and 34.6% (CI 26.5-42.7%) at 1 year. In univariate analysis, overall survival (OS) at 2 years was strongly related to stage of disease; 54% for early (CR1, chronic phase, MDS subtype-RA, good remission) versus 47% for intermediate (CR2, accelerated phase, MDS transformation, PR) versus 21% for advanced (non-remission, other subtypes of MDS) (p=0.0001). There was no impact of gender, age, diagnosis, intensity of conditioning regimen, use of serotherapy in the conditioning regimen, CBU number, TNC or CD34+ dose, HLA or ABO matching, or year of UCBT (2000-08 versus 2009-12) on OS. In a subgroup analysis of acute leukaemia, the relationship between 2 year OS and disease status was stronger; 60% for early versus 43% for intermediate versus 0% for advanced (p=0.000008), with improved survival outcomes with the use of dCBU over single unit UCBT (50% vs 34%), although this did not achieve significance (p=0.15). This retrospective national analysis supports the evolution of UCBT as an effective treatment in adults without an otherwise available donor. Outcomes are comparable to similar patient groups treated with unrelated blood and marrow transplantation. The best outcomes are achieved in early and intermediate risk disease. The benefit of national guidelines is supported by more than doubling of activity since publication in 2009 without deterioration in outcomes. Whether the national prospective clinical trials will deliver improved UK outcomes will be determined following initial analysis in 2014. Reference Shaw BE, et al. Recommendations for a standard UK approach to incorporating umbilical cord blood into clinical transplantation practice: conditioning protocols and donor selection algorithms. Bone Marrow Transplantation 2009;44:7-12 Disclosures: Gluckman: Cord use: Honoraria; gamida: Honoraria.