Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.

Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing.

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning.

T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

Ivosidenib or enasidenib combined with intensive chemotherapy in patients with newly diagnosed AML: a phase 1 study.

Ivosidenib (AG-120) and enasidenib (AG-221) are targeted oral inhibitors of the mutant isocitrate dehydrogenase (mIDH) 1 and 2 enzymes, respectively. Given their effectiveness as single agents in mIDH1/2 relapsed or refractory acute myeloid leukemia (AML), this phase 1 study evaluated the safety and efficacy of ivosidenib or enasidenib combined with intensive chemotherapy in patients with newly diagnosed mIDH1/2 AML. Ivosidenib 500 mg once daily and enasidenib 100 mg once daily were well tolerated in this setting, with safety profiles generally consistent with those of induction and consolidation chemotherapy alone. The frequency of IDH differentiation syndrome was low, as expected given the concurrent administration of cytotoxic chemotherapy. In patients receiving ivosidenib, the frequency and grades of QT interval prolongation were similar to those observed with ivosidenib monotherapy. Increases in total bilirubin were more frequently observed in patients treated with enasidenib, consistent with this inhibitor's known potential to inhibit UGT1A1, but did not appear to have significant clinical consequences. In patients receiving ivosidenib (n = 60) or enasidenib (n = 91), end-of-induction complete remission (CR) rates were 55% and 47%, respectively, and CR/CR with incomplete neutrophil or platelet recovery (CR/CRi/CRp) rates were 72% and 63%, respectively. In patients with a best overall response of CR/CRi/CRp, 16/41 (39%) receiving ivosidenib had IDH1 mutation clearance and 15/64 (23%) receiving enasidenib had IDH2 mutation clearance by digital polymerase chain reaction; furthermore, 16/20 (80%) and 10/16 (63%), respectively, became negative for measurable residual disease by multiparameter flow cytometry. This trial was registered at www.clinicaltrials.gov as #NCT02632708.

Phase 1 study of anti-CD47 monoclonal antibody CC-90002 in patients with relapsed/refractory acute myeloid leukemia and high-risk myelodysplastic syndromes.

CC-90002 is an anti-CD47 antibody that inhibits CD47-SIRPα interaction and enables macrophage-mediated killing of tumor cells in hematological cancer cell lines. In this first clinical, phase 1, dose-escalation and -expansion study (CC-90002-AML-001; NCT02641002), we evaluated CC-90002 in patients with relapsed/refractory acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS). CC-90002 was administered in escalating doses of 0.1-4.0 mg/kg, using a modified 3 + 3 design. Primary endpoints included dose-limiting toxicities (DLTs), non-tolerated dose (NTD), maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary endpoints included preliminary efficacy, pharmacokinetics, and presence/frequency of anti-drug antibodies (ADAs). Between March 2016 and July 2018, 28 patients were enrolled (24 with AML and 4 with MDS) at 6 sites across the USA. As of July 18, 2018, all patients had discontinued, mainly due to death or progressive disease. The most common treatment-emergent adverse events were diarrhea (46.4%), thrombocytopenia (39.3%), febrile neutropenia (35.7%), and aspartate aminotransferase increase (35.7%). Four patients experienced DLTs (1 patient had grade 4 disseminated intravascular coagulation and grade 5 cerebral hemorrhage, 1 had grade 3 purpura, 1 had grade 4 congestive cardiac failure and grade 5 acute respiratory failure, and another had grade 5 sepsis). The NTD and MTD were not reached. No objective responses occurred. CC-90002 serum exposure was dose-dependent. ADAs were present across all doses, and the proportion of ADA-positive patients in cycle 1 increased over time. Despite no unexpected safety findings, the CC-90002-AML-001 study was discontinued in dose escalation for lack of monotherapy activity and evidence of ADAs. However, as other anti-CD47 agents in clinical trials are showing promising early results for AML and MDS, understanding preclinical and clinical differences between individual agents in this class will be of high importance.

Pomalidomide plus low-dose dexamethasone in relapsed refractory multiple myeloma after lenalidomide treatment failure.

Patients with relapsed/refractory multiple myeloma (RRMM) for whom the benefits of lenalidomide have been exhausted in early treatment lines need effective therapies. In cohort A of the phase 2 MM-014 trial, we examined the safety and efficacy of pomalidomide plus low-dose dexamethasone immediately after lenalidomide-based treatment failure in patients with RRMM and two prior lines of therapy. Pomalidomide 4 mg was given on days 1 to 21 of 28-day cycles. Dexamethasone 40 mg (20 mg for patients aged >75 years) was given on days 1, 8, 15 and 22 of 28-day cycles. The primary endpoint was overall response rate (ORR), and secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. The intention-to-treat population comprised 56 patients; all received prior lenalidomide (87·5% lenalidomide refractory) and 39 (69·6%) received prior bortezomib. ORR was 32·1% (28·2% in the prior-bortezomib subgroup). Median PFS was 12·2 months (7·9 months in the prior-bortezomib subgroup). Median OS was 41·7 months (38·6 months in the prior-bortezomib subgroup). The most common grade 3/4 treatment-emergent adverse events were anaemia (25·0%), pneumonia (14·3%) and fatigue (14·3%). These findings support earlier sequencing of pomalidomide-based therapy in lenalidomide-pretreated patients with RRMM, including those who have become refractory to lenalidomide. Trial registration: www.ClinicalTrials.gov identifier NCT01946477.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells.

Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34+ cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2-M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation toward monocytes but elicit diverse effects on megakaryo-erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context-specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. Stem Cells 2018;36:1663-1675.

Streptococcus pneumoniae as a Cause of Mycotic and Infected Aneurysms in Patients without Respiratory Features: Challenging Diagnoses Aided by 16S PCR.

BACKGROUND: Streptococcus pneumoniae is considered a rare cause of mycotic aneurysms. The microbiological diagnosis of mycotic aneurysms can be difficult, and many patients have negative blood culture results. METHODS: We describe a series of four consecutive cases of mycotic aneurysms caused by S. pneumoniae with no respiratory features or extravascular septic foci. In two patients with negative blood culture results, 16S PCR was used for the diagnosis of S. pneumoniae infection. RESULTS: Four men with mycotic aneurysms affecting the aorta, axillary, and popliteal arteries caused by S. pneumoniae presented to our center between 2015 and 2016. All were treated with at least one month of intravenous antibiotics, followed by at least 4 weeks of oral antibiotics. Two were additionally managed using endovascular surgical techniques, and one underwent an open surgical repair. The fourth patient presented with bilateral popliteal aneurysms, one of which ruptured and was managed using surgical ligation and bypass, whereas the other side subsequently ruptured and was repaired endovascularly. Three of the four patients are currently off antibiotics and considered cured, while one died of an unrelated cause. CONCLUSIONS: S. pneumoniae should be considered a potential causative agent of mycotic aneurysms. Diagnosis can be confirmed using 16S PCR, especially in patients where peripheral blood cultures are uninformative.

Human lymphoid development in the absence of common γ-chain receptor signaling.

Despite the power of model systems to reveal basic immunologic mechanisms, critical differences exist between species that necessitate the direct study of human cells. Illustrating this point is the difference in phenotype between patients with SCID caused by mutations affecting the common γ-chain (γc) cytokine signaling pathway and mice with similar mutations. Although in both species, null mutations in either IL-2RG (which encodes γc), or its direct downstream signaling partner JAK3, result in T and NK cell deficiency, an associated B cell deficiency is seen in mice but not in humans with these genetic defects. In this study, we applied recent data that have revised our understanding of the earliest stages of lymphoid commitment in human bone marrow (BM) to determine the requirement for signaling through IL-2RG and JAK3 in normal development of human lymphoid progenitors. BM samples from SCID patients with IL-2RG (n = 3) or JAK3 deficiency (n = 2), which produce similar "T-NK-B+" clinical phenotypes, were compared with normal BM and umbilical cord blood as well as BM from children on enzyme treatment for adenosine deaminase-deficient SCID (n = 2). In both IL-2RG- and JAK3-SCID patients, the early stages of lymphoid commitment from hematopoietic stem cells were present with development of lymphoid-primed multipotent progenitors, common lymphoid progenitors and B cell progenitors, normal expression patterns of IL-7RA and TLSPR, and the DNA recombination genes DNTT and RAG1. Thus, in humans, signaling through the γc pathway is not required for prethymic lymphoid commitment or for DNA rearrangement.

Assessing Patients beyond the Simple Optics of BMI: The Concomitant Role of Sarcopenia and BMI in Predicting Kidney Transplant Outcomes.

BACKGROUND: Body composition is associated with prognosis in many clinical settings, and patients undergoing kidney transplantation are often high risk with multiple comorbidities. We aimed to assess the effect of sarcopenia and body composition on transplant outcomes. METHODS: We performed a retrospective analysis of 274 kidney transplants with CT scans within 3 years of transplantation. The skeletal muscle index (SMI) at the L3 vertebrae was used to evaluate sarcopenia (SMI < 40.31 cm2/m2 in males, <30.88 cm2/m2 in females). Sarcopenia, body mass index (BMI), and the visceral-to-subcutaneous-fat ratio (VSR) were assessed separately. We also used a composite BMI/sarcopenia measurement in four patient groups: BMI < 25/Non-Sarcopenic, BMI < 25/Sarcopenic, BMI > 25/Non-Sarcopenic, and BMI > 25/Sarcopenic. The outcomes measured were eGFR (1 and 3 months; and 1, 3, and 5 years), delayed graft function (DGF), rejection, major adverse cardiovascular events (MACE), and post-operative complications. RESULTS: Sarcopenia was associated with an increased 1-year risk of MACE (OR 3.41, p = 0.036). BMI alone had no effect on function, DGF, MACE, or on other complications. High VSR was associated with a lower risk of DGF (OR 0.473, p = 0.016). When sarcopenia and BMI were assessed together, the BMI > 25/sarcopenic patients had the poorest outcomes, with increased risk of MACE (OR 26.06, p = 0.001); poorer eGFR at 1, 3, 12, and 36 months; (p < 0.05 at all timepoints), and poorer graft survival (p = 0.002). CONCLUSIONS: Sarcopenia alone is associated with an increased risk of MACE. Overweight sarcopenic patients are additionally at increased risk of graft loss and have poorer graft function for up to three years.

Distinct roles of hematopoietic cytokines in the regulation of leukemia stem cells in murine MLL-AF9 leukemia.

Lymphoid-primed multipotent progenitor (LMPP)-like and granulocyte-monocyte progenitor (GMP)-like leukemia stem cells (LSCs) co-exist in the blood of most patients with acute myeloid leukemia (AML). Complete elimination of both types of LSCs is required to cure AML. Using an MLL-AF9-induced murine AML model, we studied the role of hematopoietic cytokines in the survival of LMPP- and GMP-like LSCs. We found that SCF or FLT3L promotes the survival of LMPP-like LSCs by stimulating Stat5-mediated Mcl1 expression, whereas interleukin-3 (IL-3) or IL-6 induces the survival of GMP-like LSCs by stimulating Stat3/nuclear factor κB (NF-κB)-mediated Bcl2 expression. Functional study demonstrated that, compared to AML cells cultured in IL-3 and IL-6 medium, AML cells in SCF- or Flt3L-only culture are highly clonogenic in in vitro culture and are highly leukemogenic in vivo. Our study suggests that co-inhibition of both STAT5-MCL1 and STAT3/NF-κB-BCL2 signaling might represent an improved treatment strategy against AML, specifically AML cases with a monocytic phenotype and/or FLT3 mutations.

Pomalidomide/Daratumumab/Dexamethasone in Relapsed or Refractory Multiple Myeloma: Final Overall Survival From MM-014.

BACKGROUND: Patients with relapsed or refractory multiple myeloma (RRMM) who have exhausted lenalidomide benefits require improved therapies. The 3-cohort phase 2 MM-014 trial (NCT01946477) explored pomalidomide in early lines of treatment for lenalidomide-exposed RRMM. In cohort B, pomalidomide plus daratumumab and dexamethasone (DPd) showed promising efficacy (median follow-up 28.4 months), as previously reported. Here, we report final overall survival (OS) in cohort B. METHODS: Patients aged ≥ 18 years were treated in 28-day cycles: pomalidomide 4 mg orally daily from days 1 to 21; daratumumab 16 mg/kg intravenously on days 1, 8, 15, and 22 (cycles 1-2), days 1 and 15 (cycles 3-6), and day 1 (cycle ≥ 7); and dexamethasone 40 mg (age ≤ 75 years) or 20 mg (age > 75 years) orally on days 1, 8, 15, and 22. The primary endpoint was ORR. OS and safety were secondary endpoints. RESULTS: Among 112 patients enrolled, 85 (75.9%) had lenalidomide-refractory disease and 27 (24.1%) had lenalidomide-relapsed disease. At a median follow-up of 41.9 months (range, 0.4-73.1), median OS was 56.7 months (95% confidence interval, 46.5-not reached). Treatment-emergent adverse events related to, and leading to discontinuation of, pomalidomide, dexamethasone, or daratumumab occurred in 7 (6.3%), 9 (8.0%), and 6 (5.4%) patients, respectively. CONCLUSION: With long-term follow-up, our results show favorable OS with DPd. The safety profile was consistent with previous reports, with no new safety signals identified. IMiD agent-based therapy can still be considered in patients with RRMM who experience progressive disease on or after lenalidomide.

Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method.

Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using 'off-the-shelf' products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

The management of dialysis access thrombosis during the COVID-19 pandemic.

BACKGROUND: Maintaining patent access is essential for haemodialysis dependent end stage renal failure patients. The COVID-19 pandemic has significantly affected surgical and interventional radiology services worldwide. We aimed to review the impact COVID-19 has caused to the management of acute dialysis access thrombosis. METHODS: We conducted a single centre retrospective review of outcomes of patients with arteriovenous fistula and arteriovenous graft thrombosis between March and May 2020, which coincided with the first peak of the COVID-19 pandemic in London, and a similar period in the previous year, March-May 2019. Outcomes in both cohorts of patients were compared, including attempts at salvage, salvage success, 1-month patency rates after salvage and subsequent surgery on the same access. We also analysed the use of tunnelled haemodialysis lines (THL), either due to failed salvage attempts or when salvage was not attempted. RESULTS: There was a similar incidence of access thrombosis in both periods (26 cases in 2019, 38 in 2020). There were 601 patients dialysing via an arteriovenous fistula or graft in 2019, and 568 patients in 2020. Access salvage, when attempted, had similar success rates and 1-month patency (salvage success 74% vs 80%, p = 0.39; 1-month patency 55% vs 62%, p = 0.69). The proportion of patients where access salvage was not attempted and a THL inserted was significantly higher in 2020 compared to 2019 (32% vs 4%, p = 0.007). There were more patients who subsequently had surgery to salvage or revise the same access in 2019 compared to 2020 (62% vs 13%, p < 0.001). CONCLUSIONS: During the peak of the COVID-19 pandemic, there were fewer attempts at access salvage. This was a conscious decision due to increased pressure on the healthcare system, access to emergency interventional radiology or operative theatres and the perceived risk/benefit ratio of access salvage. The long-term effects of this change in practice remain unknown.

Transient SARS-CoV-2 RNA-Dependent RNA Polymerase Mutations after Remdesivir Treatment for Chronic COVID-19 in Two Transplant Recipients: Case Report and Intra-Host Viral Genomic Investigation.

Remdesivir is the first FDA-approved drug for treating severe SARS-CoV-2 infection and targets RNA-dependent RNA polymerase (RdRp) that is required for viral replication. To monitor for the development of mutations that may result in remdesivir resistance during prolonged treatment, we sequenced SARS-CoV-2 specimens collected at different treatment time points in two transplant patients with severe COVID-19. In the first patient, an allogeneic hematopoietic stem cell transplant recipient, a transient RdRp catalytic subunit mutation (nsp12:A449V) was observed that has not previously been associated with remdesivir resistance. As no in vitro study had been conducted to elucidate the phenotypic effect of nsp12:A449V, its clinical significance is unclear. In the second patient, two other transient RdRp mutations were detected: one in the catalytic subunit (nsp12:V166A) and the other in an accessory subunit important for processivity (nsp7:D67N). This is the first case report for a potential link between the nsp12:V166A mutation and remdesivir resistance in vivo, which had only been previously described by in vitro studies. The nsp7:D67N mutation has not previously been associated with remdesivir resistance, and whether it has a phenotypic effect is unknown. Our study revealed SARS-CoV-2 genetic dynamics during remdesivir treatment in transplant recipients that involved mutations in the RdRp complex (nsp7 and nsp12), which may be the result of selective pressure. These results suggest that close monitoring for potential resistance during the course of remdesivir treatment in highly vulnerable patient populations may be beneficial. Development and utilization of diagnostic RdRp genotyping tests may be a future direction for improving the management of chronic COVID-19.

Generation of antigen-specific mature T cells from RAG1-/-RAG2-/-B2M-/- stem cells by engineering their microenvironment.

Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1-/-RAG2-/-B2M-/- human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.