Anti-T-lymphocyte globulin exposure is associated with acute graft-versus-host disease and relapse in pediatric acute lymphoblastic leukemia patients undergoing hematopoietic stem cell transplantation: a multinational prospective study.

Anti T-lymphocyte globulin (ATLG) is used in hematopoietic stem cell transplantation (HSCT) to prevent graft-versus-host disease (GvHD) and graft failure. To date, insight in ATLG pharmacokinetics and -dynamics (PK/PD) is limited, and population PK (POPPK) models are lacking. In this prospective study, we describe ATLG POPPK using NONMEM® and the impact of ATLG exposure on clinical outcome and immune reconstitution in a homogeneous cohort of pediatric acute lymphoblastic leukemia (ALL) patients transplanted with a matched unrelated donor and receiving uniform ATLG dosing. Based on 121 patients and 812 samples for POPPK analysis, a two-compartmental model with parallel linear and non-linear clearance and bodyweight as covariate, best described the ATLG concentrationtime data. The level of ATLG exposure (day active ATLG day 16 8.2%, p.

Outcomes of HLA-mismatched HSCT with TCRαß/CD19 depletion or post-HSCT cyclophosphamide for inborn errors of immunity.

HLA-mismatched transplants with either in vitro depletion of CD3+TCRαß/CD19 (TCRαß) cells or in vivo T-cell depletion using post-transplant cyclophosphamide (PTCY) have been increasingly used for patients with inborn errors of immunity (IEI). We performed a retrospective multicenter study via the EBMT registry on 306 children with IEI undergoing first transplant between 2010-2019 from an HLA-mismatched donor using TCRαß (n=167) or PTCY (n=139). Median age at HSCT was 1.2 years (range, 0.03-19.6 years). The 3-year overall survival (OS) was 78% (95% confidence interval (CI), 71-84%) after TCRαß and 66% (57-74%) after PTCY (p=0.013). Pre-HSCT morbidity score (hazard ratio (HR) 2.27, 1.07-4.80, p=0.032) and non-Busulfan/Treosulfan conditioning (HR 3.12, 1.98-4.92, p<0.001) were the only independent predictors of unfavorable OS. The 3-year event-free survival (EFS) was 58% (50-66%) after TCRαß and 57% (48-66%) after PTCY (p=0.804). Cumulative incidence of severe acute GvHD was higher after PTCY (15%, 9-21%) than TCRαß (6%, 2-9%, p=0.007), with no difference in chronic GvHD (PTCY, 11%, 6-17%; TCRαß, 7%, 3-11%, p=0.173). The 3-year GvHD-free EFS was 53% (44-61%) after TCRαß and 41% (32-50%) after PTCY (p=0.080). PTCY had significantly higher rates of veno-occlusive disease (14.4% versus TCRαß 4.9%, p=0.009), acute kidney injury (12.7% versus 4.6%, p=0.032) and pulmonary complications (38.2% versus 24.1%, p=0.017). Adenoviraemia (18.3% versus PTCY 8.0%, p=0.015), primary graft failure (10%, versus 5%, p=0.048), and second HSCT (17.4% versus 7.9%, p=0.023) were significantly higher in TCRαß. In conclusion, this study demonstrates that both approaches are suitable options in IEI patients, although characterized by different advantages and outcomes.

Busulfan-fludarabine- or treosulfan-fludarabine-based conditioning before allogeneic HSCT from matched sibling donors in paediatric patients with sickle cell disease: A study on behalf of the EBMT Paediatric Diseases and Inborn Errors Working Parties.

How important is choice of conditioning regimen in allogeneic haematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD)? We compared HSCT outcomes by conditioning regimen in paediatric patients with SCD from the EBMT registry. In 2010-2020, 251 patients aged <18 years underwent a first matched sibling donor (MSD) HSCT with conditioning based on busulfan-fludarabine (bu-flu; n = 89) or treosulfan-fludarabine (treo-flu; n = 162). In the bu-flu and treo-flu groups, 51.7% and 99.4% of patients, respectively, received thiotepa. Median follow-up was 2.7 years. Two-year overall survival (OS) was 98.7% (95% confidence interval [CI]: 90.9-99.8) with bu-flu and 99.3% (95% CI: 95.2-99.9) with treo-flu (p = 0.63). Grade III-IV acute graft-versus-host disease (GVHD) at 100 days was 2.4% (95% CI: 0.4-7.5) and 0.6% (0.1%-3.2%) for bu-flu and treo-flu respectively (p = 0.25). The 2-year incidence of extensive chronic GVHD was 1.5% (95% CI: 0.1-7.3) with bu-flu and 8.0% (95% CI: 4.1-13.3) with treo-flu (p = 0.057). These multinational data confirm the excellent curative capacity of MSD HSCT with myeloablative conditioning. Both conditioning regimens yielded excellent OS, low rates of acute and chronic GVHD, and low rates of graft failure.

JAK inhibitor treatment for inborn errors of JAK/STAT signaling: An ESID/EBMT-IEWP retrospective study.

BACKGROUND: Inborn errors of immunity (IEI) with dysregulated JAK/STAT signaling present with variable manifestations of immune dysregulation and infections. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but initially reported outcomes were poor. JAK inhibitors (JAKi) offer a targeted treatment option that may be an alternative or bridge to HSCT. However, data on their current use, treatment efficacy and adverse events are limited. OBJECTIVE: We evaluated the current off-label JAKi treatment experience for JAK/STAT inborn errors of immunity (IEI) among European Society for Immunodeficiencies (ESID)/European Society for Blood and Marrow Transplantation (EBMT) Inborn Errors Working Party (IEWP) centers. METHODS: We conducted a multicenter retrospective study on patients with a genetic disorder of hyperactive JAK/STAT signaling who received JAKi treatment for at least 3 months. RESULTS: Sixty-nine patients (72% children) were evaluated (45 STAT1 gain of function [GOF], 21 STAT3-GOF, 1 STAT5B-GOF, 1 suppressor of cytokine signaling 1 [aka SOCS1] loss of function, 1 JAK1-GOF). Ruxolitinib was the predominantly prescribed JAKi (80%). Overall, treatment resulted in improvement (partial or complete remission) of clinical symptoms in 87% of STAT1-GOF and in 90% of STAT3-GOF patients. We documented highly heterogeneous dosing and monitoring regimens. The response rate and time to response varied across different diseases and manifestations. Adverse events including infection and weight gain were frequent (38% of patients) but were mild (grade I-II) and transient in most patients. At last follow-up, 52 (74%) of 69 patients were still receiving JAKi treatment, and 11 patients eventually underwent HSCT after receipt of previous JAKi bridging therapy, with 91% overall survival. CONCLUSIONS: Our study suggests that JAKi may be highly effective to treat symptomatic JAK/STAT IEI patients. Prospective studies to define optimal JAKi dosing for the variable clinical presentations and age ranges should be pursued.

The value of using patient-reported outcomes for health screening during long-term follow-up after paediatric stem cell transplantation for nonmalignant diseases.

INTRODUCTION: The assessment of using patient-reported outcomes (PROs) within comprehensive care follow-up programmes, specifically focused on health screening, remains largely unexplored. PROs were implemented in our late effects and comprehensive care programme after paediatric hematopoietic stem cell transplantation (HSCT) for nonmalignant diseases. The programme focuses solely on screening of physical and mental health and on discussing PROs during the consultation. METHODS: The primary method of this study was semistructured interviews to explore the perspective of both patients and healthcare providers' (HCP) on the use of PROs, which were thematically analyzed. Additionally, an explorative quantitative approach with patient-reported experience measures (PREMS) was used, with a pretest-posttest design, to assess whether the use of PROs was accompanied by more patient-centred care. RESULTS: From the patient-interviews (N = 15) four themes were extracted: use of PROs (1) help to discuss topics; (2) make the patients feel understood; (3) create a moment of self-reflection; and (4) make consultations more efficient. Pre- and postimplementation analysis of PREMs (N = 40) did not show significant differences in terms of patient-centeredness. CONCLUSION: Our results demonstrate the added value of integrating PROs for health screening purposes within the long-term follow-up programme after paediatric HSCT, as perceived by both patient and HCP. With the active use of PROs, patients are stimulated to consciously assess their health status. PATIENT CONTRIBUTION: This study included patients as participants. Caregivers were approached if patients were below a certain age. Additionally, preliminary results were shared with all patients (including nonparticipants) during a patient conference day.

Persistent Hypogammaglobulinemia after Receiving Rituximab Post-HSCT Is Not Caused by an Intrinsic B Cell Defect.

In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naïve B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers.

Pediatric Bone Marrow Failure: A Broad Landscape in Need of Personalized Management.

Irreversible severe bone marrow failure (BMF) is a life-threatening condition in pediatric patients. Most important causes are inherited bone marrow failure syndromes (IBMFSs) and (pre)malignant diseases, such as myelodysplastic syndrome (MDS) and (idiopathic) aplastic anemia (AA). Timely treatment is essential to prevent infections and bleeding complications and increase overall survival (OS). Allogeneic hematopoietic stem cell transplantation (HSCT) provides a cure for most types of BMF but cannot restore non-hematological defects. When using a matched sibling donor (MSD) or a matched unrelated donor (MUD), the OS after HSCT ranges between 60 and 90%. Due to the introduction of post-transplantation cyclophosphamide (PT-Cy) to prevent graft versus host disease (GVHD), alternative donor HSCT can reach similar survival rates. Although HSCT can restore ineffective hematopoiesis, it is not always used as a first-line therapy due to the severe risks associated with HSCT. Therefore, depending on the underlying cause, other treatment options might be preferred. Finally, for IBMFSs with an identified genetic etiology, gene therapy might provide a novel treatment strategy as it could bypass certain limitations of HSCT. However, gene therapy for most IBMFSs is still in its infancy. This review summarizes current clinical practices for pediatric BMF, including HSCT as well as other disease-specific treatment options.

Advances in gene therapy for inborn errors of immunity.

PURPOSE OF REVIEW: Provide an overview of the landmark accomplishments and state of the art of gene therapy for inborn errors of immunity (IEI). RECENT FINDINGS: Three decades after the first clinical application of gene therapy for IEI, there is one market authorized product available, while for several others efficacy has been demonstrated or is currently being tested in ongoing clinical trials. Gene editing approaches using programmable nucleases are being explored preclinically and could be beneficial for genes requiring tightly regulated expression, gain-of-function mutations and dominant-negative mutations. SUMMARY: Gene therapy by modifying autologous hematopoietic stem cells (HSCs) offers an attractive alternative to allogeneic hematopoietic stem cell transplantation (HSCT), the current standard of care to treat severe IEI. This approach does not require availability of a suitable allogeneic donor and eliminates the risk of graft versus host disease (GvHD). Gene therapy can be attempted by using a viral vector to add a copy of the therapeutic gene (viral gene addition) or by using programmable nucleases (gene editing) to precisely correct mutations, disrupt a gene or introduce an entire copy of a gene at a specific locus. However, gene therapy comes with its own challenges such as safety, therapeutic effectiveness and access. For viral gene addition, a major safety concern is vector-related insertional mutagenesis, although this has been greatly reduced with the introduction of safer vectors. For gene editing, the risk of off-site mutagenesis is a main driver behind the ongoing search for modified nucleases. For both approaches, HSCs have to be manipulated ex vivo, and doing this efficiently without losing stemness remains a challenge, especially for gene editing.

Predicting Patient Death after Allogeneic Stem Cell Transplantation for Inborn Errors Using Machine Learning (PREPAD): A European Society for Blood and Marrow Transplantation Inborn Errors Working Party Study.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many inborn errors of immunity, metabolism, and hematopoiesis. No predictive models are available for these disorders. We created a machine learning model using XGBoost to predict survival after HSCT using European Society for Blood and Marrow Transplant registry data of 10,888 patients who underwent HSCT for inborn errors between 2006 and 2018, and compared it to a simple linear Cox model, an elastic net Cox model, and a random forest model. The XGBoost model had a cross-validated area under the curve value of .73 at 1 year, which was significantly superior to the other models, and it accurately predicted for countries excluded while training. It predicted close to 0% and >30% mortality more often than other models at 1 year, while maintaining good calibration. The 5-year survival was 94.7% in the 25% of patients at lowest risk and 62.3% in the 25% at highest risk. Within disease and donor subgroups, XGBoost outperformed the best univariate predictor. We visualized the effect of the main predictors-diagnosis, performance score, patient age and donor type-using the SHAP ML explainer and developed a stand-alone application, which can predict using the model and visualize predictions. The risk of mortality after HSCT for inborn errors can be accurately predicted using an explainable machine learning model. This exceeds the performance of models described in the literature. Doing so can help detect deviations from expected survival and improve risk stratification in trials.

Long-term parental distress after pediatric hematopoietic stem cell transplantation for nonmalignant diseases.

BACKGROUND: Survival rates have continued to increase for pediatric hematopoietic stem cell transplantation (HSCT) for nonmalignant diseases. Despite the crucial role of caregivers in this high-intensity treatment, knowledge about long-term parental impact is lacking. PROCEDURE: This cross-sectional study assessed parental distress and everyday problems in parents of patients 2 years and older after pediatric HSCT for a nonmalignant disease using Distress Thermometer for Parents (DT-P), and compared outcomes to matched Dutch parents of healthy children and Dutch parents of children with a chronic condition (CC). RESULTS: Median follow-up was 5.3 years (interquartile range [IQR]: 2.9-8.6). Underlying diseases were inborn errors of immunity (N = 30), hemoglobinopathies (N = 13), and bone marrow failure (N = 27). Mothers of pediatric HSCT recipients (N = 70) reported comparable overall distress levels to mothers of healthy children, but experienced more distress related to parenting problems, specifically managing their child's emotions, discussing disease consequences, and fostering independence. Fathers of HSCT recipients (N = 45) reported higher overall distress levels and had more emotional distress compared to fathers of healthy children. CONCLUSIONS: Overall, parental distress and everyday problems of parents of HSCT recipients are comparable to those of parents of children with CC. However, there is ongoing parental burden, both emotional and in parenting, long-term after HSCT compared to parents of healthy children, and the type of burden differs between mothers and fathers. These results indicate that individualized parental supportive care should not remain restricted to the acute hospitalization phase, but also be actively offered during long-term follow-up after pediatric HSCT.

Exposure-response analysis of alemtuzumab in pediatric allogeneic HSCT for nonmalignant diseases: the ARTIC study.

Alemtuzumab (anti-CD52 antibody) is frequently prescribed to children with nonmalignant diseases undergoing allogeneic hematopoietic stem cell transplantation (HSCT) to prevent graft failure (GF) and acute graft-versus-host disease (aGVHD). The aim of this multicenter study was the characterization of alemtuzumab population pharmacokinetics to perform a novel model-based exposure-response analysis in 53 children with nonmalignant immunological or hematological disease and a median age of 4.4 years (interquartile range [IQR], 0.8-8.7). The median cumulative alemtuzumab dose was 0.6 mg/kg (IQR, 0.6-1) administered over 2 to 7 days. A 2-compartment population pharmacokinetics model with parallel linear and nonlinear elimination including allometrically scaled bodyweight (median, 17.50 kg; IQR, 8.76-33.00) and lymphocyte count at baseline (mean, 2.24 × 109/L; standard deviation ± 1.87) as significant pharmacokinetic predictors was developed using nonlinear mixed effects modeling. Based on the model-estimated median concentration at day of HSCT (0.77 µg/mL; IQR, 0.33-1.82), patients were grouped into a low- (≤0.77 µg/mL) or high- (>0.77 µg/mL) exposure groups. High alemtuzumab exposure at day of HSCT correlated with delayed CD4+ and CD8+ T-cell reconstitution (P value < .0001) and increased risk of GF (P value = .043). In contrast, alemtuzumab exposure did not significantly influence the incidence of aGVHD grade ≥2, mortality, chimerism at 1 year, viral reactivations, and autoimmunity at a median follow-up of 3.3 years (IQR, 2.5-8.0). In conclusion, this novel population pharmacokinetics model is suitable for individualized intravenous precision dosing to predict alemtuzumab exposure in pediatric allogeneic HSCT for nonmalignant diseases, aiming at the achievement of early T-cell reconstitution and prevention of GF in future prospective studies.

Automating outcome analysis after stem cell transplantation: The YORT tool.

Hematopoietic stem cell transplantation is a high-risk procedure. Auditing and yearly outcome reviews help keep optimal quality of care and come with increased survival, but also has significant recurring costs. When data has been entered in a standardized registry, outcome analyses can be automated, which reduces work and increases standardization of performed analyses. To achieve this, we created the Yearly Outcome Review Tool (YORT), an offline, graphical tool that gets data from a single center EBMT registry export, allows the user to define filters and groups, and performs standardized analyses for overall survival, event-free survival, engraftment, relapse rate and non-relapse mortality, complications including acute and chronic Graft vs Host Disease (GvHD), and data completeness. YORT allows users to export data as analyzed to allow you to check data and perform manual analyses. We show the use of this tool on a two-year single-center pediatric cohort, demonstrating how the results for both overall and event-free survival and engraftment can be visualized. The current work demonstrates that using registry data, standardized tools can be made to analyze this data, which allows users to perform outcome reviews for local and accreditation purposes graphically with minimal effort, and help perform detailed standardized analyses. The tool is extensible to be able to accommodate future changes in outcome review and center-specific extensions.

IL-7 receptor signaling drives human B-cell progenitor differentiation and expansion.

Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor α chain (IL-7Rα) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7Rα deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7Rα deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis.

Effect of Busulfan and Treosulfan on Gonadal Function after Allogeneic Stem Cell Transplantation in Children and Adolescents with Nonmalignant Diseases Is Not Exposure-Dependent.

With an increasing number of young patients surviving into adulthood after hematopoietic stem cell transplantation (HSCT), gonadal dysfunction becomes an important late effect with significant impact on quality of life. In this retrospective study, we evaluated the exposure of busulfan (Bu) and treosulfan (Treo) in relation to gonadal function in pediatric patients who underwent HSCT for a nonmalignant disease between 1997 and 2018. In the Bu group, 56 patients could be evaluated, and gonadal dysfunction was found in 35 (63%). Lower Bu exposure (ie, cumulative area under the curve [AUC] <70 mg*h/L) was not associated with a reduced risk of gonadal dysfunction (odds ratio [OR], .92; 95% confidence interval [CI], .25 to 3.49; P = .90). In the Treo cohort, 32 patients were evaluable and gonadal insufficiency occurred in 9 patients (28%). Lower Treo exposure (AUC <1750 mg*h/L on day 1) was not associated with a reduced risk of gonadal dysfunction (OR, 1.6; 95% CI, .16 to 36.6; P = .71). Our data do not support the premise that reduced-intensity Bu-based conditioning reduces the risk for gonadal toxicity, and it is unlikely that therapeutic drug monitoring-based reduced treosulfan exposure will further limit the risk of gonadal dysfunction.

Late Effects in Pediatric Allogeneic Hematopoietic Stem Cell Transplantation for Nonmalignant Diseases: Proxy- and Patient-Reported Outcomes.

Survival rates in pediatric hematopoietic stem cell transplantation (HSCT) for nonmalignant diseases have improved due to advances in conditioning regimens, donor selection, and prophylaxis and treatment of infections and graft-versus-host disease. Insight into the long-term patient-reported outcomes (PROs) after pediatric HSCT for nonmalignant disease is lacking but essential for optimal shared decision making, counseling, and quality of care. The purpose of this research was to determine long-term patient-reported outcomes in allogeneic pediatric HSCT for nonmalignant diseases and to compare these results with Dutch reference data. This single-center cohort study evaluated PROs (PedsQL 4.0, PROMIS item banks), self- or proxy-reported, among patients at ≥2 years after pediatric allogeneic HSCT for nonmalignant disease. Mean scores were compared with those of the Dutch general population. Of 171 eligible patients, 119 participated, for a 70% response rate. The median patient age was 15.8 years (range, 2 to 49 years), and the median duration of follow-up was 8.7 years (range, 2 to 34 years). Indications for HSCT included inborn errors of immunity (n = 41), hemoglobinopathies (n = 37), and bone marrow failure (n = 41). Compared with reference data, significantly lower scores were found in adolescents (age 13 to 17 years) on the Total, Physical Health, and School Functioning PedsQL subscales. Significantly more Sleep Disturbance was reported in children (age 8 to 18 years). On the other hand, significantly better scores were seen on PROMIS Fatigue (age 5 to 7 years) and Pain Interference (age 8 to 18 years) and, in adults (age 19 to 30 years), on Depressive Symptoms and Sleep Disturbance. This study showed better or comparable very long-term PROs in patients after pediatric HSCT for nonmalignant diseases compared with the reference population. Children and adolescents seem to be the most affected, indicating the need for supportive care to prevent impaired quality of life and, more importantly, to amplify their long-term well-being.

A unique immune signature in blood separates therapy-refractory from therapy-responsive acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is an immune cell‒driven, potentially lethal complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. We applied mass cytometry (CyTOF) to dissect circulating myeloid and lymphoid cells in children with severe (grade III-IV) aGVHD treated with immune suppressive drugs alone (first-line therapy) or in combination with mesenchymal stromal cells (MSCs; second-line therapy). These results were compared with CyTOF data generated in children who underwent transplantation with no aGVHD or age-matched healthy control participants. Onset of aGVHD was associated with the appearance of CD11b+CD163+ myeloid cells in the blood and accumulation in the skin and GI tract. Distinct T-cell populations, including TCRγδ+ cells, expressing activation markers and chemokine receptors guiding homing to the skin and GI tract were found in the same blood samples. CXCR3+ T cells released inflammation-promoting factors after overnight stimulation. These results indicate that lymphoid and myeloid compartments are triggered at aGVHD onset. Immunoglobulin M (IgM) presumably class switched, plasmablasts, and 2 distinct CD11b- dendritic cell subsets were other prominent immune populations found early during the course of aGVHD in patients refractory to both first- and second-line (MSC-based) therapy. In these nonresponding patients, effector and regulatory T cells with skin- or gut-homing receptors also remained proportionally high over time, whereas their frequencies declined in therapy responders. Our results underscore the additive value of high-dimensional immune cell profiling for clinical response evaluation, which may assist timely decision-making in the management of severe aGVHD.

Development and Validation of an Efficient and Highly Sensitive Enzyme-Linked Immunosorbent Assay for Alemtuzumab Quantification in Human Serum and Plasma.

BACKGROUND: Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma. METHODS: This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation. RESULTS: The assay sensitivity (lower limit of quantification) is 0.5 ng·mL -1 , and the dynamic range is 0.78-25 ng·mL -1 . To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg·mL -1 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. CONCLUSIONS: The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

Single-cell transcriptomics in bone marrow delineates CD56dimGranzymeK+ subset as intermediate stage in NK cell differentiation.

Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56brightCD16+, CD56dimCD16+ and CD69+CXCR6+ lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56dimGzmK+ subset was identified that shared features with CD56bright and CD56dimGzmK- NK cells based on transcriptome, phenotype (NKG2AhighCD16lowKLRG1highTIGIThigh) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56bright, CD56dimGzmK+ and CD56dimGzmK- cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34+ progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells.