Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

PMCID: PMC3526610This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Impact of T‐cell depletion strategies on outcomes following hematopoietic stem cell transplantation for idiopathic aplastic anemia: A study on behalf of the European blood and marrow transplant severe aplastic anemia working party

We retrospectively analyzed the outcomes of 1837 adults and children with severe aplastic anemia (SAA) who underwent matched sibling donor (MSD) and matched unrelated donor (MUD) hemopoietic stem cell transplantation (HSCT) between 2000 and 2013. Patients were grouped by transplant conditioning containing either anti‐thymocyte globulin (ATG) (n = 1283), alemtuzumab (n = 261), or no serotherapy (NS) (n = 293). The risks of chronic GvHD were significantly reduced when ATG or alemtuzumab were compared with NS (P = .021 and .003, respectively). Acute GVHD was significantly reduced in favor of alemtuzumab compared with ATG (P = .012) and NS (P < .001). By multivariate analysis, when compared with ATG, alemtuzumab was associated with a lower risk of developing acute (OR 0.262; 95% CI 0.14‐0.47; P < .001) and chronic GVHD (HR 0.58; 95% CI 0.35‐0.94; P = .027). OS was significantly better in ATG and alemtuzumab patients compared with NS (P = .010 and .025). Our data shows inclusion of serotherapy in MSD and MUD HSCT for patients with SAA reduces chronic GVHD and provides a survival advantage over patients not receiving serotherapy. Notably, alemtuzumab reduced the risk of acute and chronic GvHD compared with ATG and indicates that alemtuzumab might be the serotherapy of choice for MSD and MUD transplants for SAA

Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)1,2,3,4,5, but toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, limits broader application. Moreover, 40–60% of patients relapse owing to poor CAR T cell persistence or emergence of CD19− clones. Some factors, including the choice of single-chain spacer6 and extracellular7 and costimulatory domains8, have a profound effect on CAR T cell function and persistence. However, little is known about the impact of CAR binding affinity. There is evidence of a ceiling above which increased immunoreceptor affinity may adversely affect T cell responses9,10,11. We generated a novel CD19 CAR (CAT) with a lower affinity than FMC63, the high-affinity binder used in many clinical studies1,2,3,4. CAT CAR T cells showed increased proliferation and cytotoxicity in vitro and had enhanced proliferative and in vivo antitumor activity compared with FMC63 CAR T cells. In a clinical study (CARPALL, NCT02443831), 12/14 patients with relapsed/refractory pediatric B cell acute lymphoblastic leukemia treated with CAT CAR T cells achieved molecular remission. Persistence was demonstrated in 11 of 14 patients at last follow-up, with enhanced CAR T cell expansion compared with published data. Toxicity was low, with no severe CRS. One-year overall and event-free survival were 63% and 46%, respectivel

Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode <it>Caenorhabditis elegans</it>, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from <it>C. elegans</it>.</p> <p>Results</p> <p>We report improved and effective <it>in vitro </it>RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, <it>Brugia malayi</it>. The cellular disorganization observed in <it>B. malayi </it>embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their <it>C. elegans </it>orthologs. Targeting the <it>B. malayi </it>cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in <it>C. elegans</it>. Cellular phenotypes induced by our <it>in vitro </it>RNAi procedure can be observed by immunofluorescence in as little as one week.</p> <p>Conclusions</p> <p>We observed cytological defects following RNAi targeting all seven <it>B. malayi </it>transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism <it>C. elegans</it>. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.</p

Untargeted proteomics enables ultra-rapid variant prioritisation in mitochondrial and other rare diseases

\ua9 The Author(s) 2025.Background: Only half of individuals with suspected rare diseases receive a genetic diagnosis following genomic testing. A genetic diagnosis allows access to appropriate care, restores reproductive confidence and reduces the number of potentially unnecessary interventions. A major barrier is the lack of disease agnostic functional tests suitable for implementation in routine diagnostics that can provide evidence supporting pathogenicity of novel variants, especially those refractory to RNA sequencing. Methods: Focusing on mitochondrial disease, we describe an untargeted mass-spectrometry based proteomics pipeline that can quantify proteins encoded by &gt; 50% of Mendelian disease genes and &gt; 80% of known mitochondrial disease genes in clinically relevant sample types, including peripheral blood mononuclear cells (PBMCs). In total we profiled &gt; 90 individuals including undiagnosed individuals suspected of mitochondrial disease and a supporting cohort of disease controls harbouring pathogenic variants in nuclear and mitochondrial genes. Proteomics data were benchmarked against pathology accredited respiratory chain enzymology to assess the performance of proteomics as a functional test. Proteomics testing was subsequently applied to individuals with suspected mitochondrial disease, including a critically ill infant with a view toward rapid interpretation of variants identified in ultra-rapid genome sequencing. Results: Proteomics testing provided evidence to support variant pathogenicity in 83% of individuals in a cohort with confirmed mitochondrial disease, outperforming clinical respiratory chain enzymology. Freely available bioinformatic tools and criteria developed for this study (https://rdms.app/) allow mitochondrial dysfunction to be identified in proteomics data with high confidence. Application of proteomics to undiagnosed individuals led to 6 additional diagnoses, including a mitochondrial phenocopy disorder, highlighting the disease agnostic nature of proteomics. Use of PBMCs as a sample type allowed rapid return of proteomics data supporting pathogenicity of novel variants identified through ultra-rapid genome sequencing in as little as 54 h. Conclusions: This study provides a framework to support the integration of a single untargeted proteomics test into routine diagnostic practice for the diagnosis of mitochondrial and potentially other rare genetic disorders in clinically actionable timelines, offering a paradigm shift for the functional validation of genetic variants

Severe Aplastic Anemia and PNH

Severe aplastic anemia (SAA) is an autoimmune disorder (AID) due to the attack of autoreactive cytotoxic T lymphocytes to the hematopoietic component of the bone marrow