During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including 3 rd dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is less after infection compared to all vaccines evaluated, but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks post-vaccination in some cases. SARS-CoV-2 antibody specificity, breadth and maturation are affected by imprinting from exposure history, and distinct histological and antigenic contexts in infection compared to vaccination.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type–specific RNA splicing was discovered and analyzed across tissues within an individual.

Duchenne muscular dystrophy (DMD) causes profound and progressive muscle weakness and loss, resulting in early death. DMD is usually caused by frameshifting deletions in the gene DMD, which leads to absence of dystrophin protein. Dystrophin binds to F-actin and components of the dystrophin-associated glycoprotein complex and protects the sarcolemma from contraction-induced injury. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of an intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans by this method. We performed a small-molecule screen to identify existing drugs that enhance antisense-directed exon skipping. We found that dantrolene, currently used to treat malignant hyperthermia, potentiates antisense oligomer-guided exon skipping to increase exon skipping to restore the mRNA reading frame, the sarcolemmal dystrophin protein, and the dystrophin glycoprotein complex in skeletal muscles of mdx mice when delivered intramuscularly or intravenously. Further, dantrolene synergized with multiple weekly injections of antisense to increase muscle strength and reduce serum creatine kinase in mdx mice. Dantrolene similarly promoted antisense-mediated exon skipping in reprogrammed myotubes from DMD patients. Ryanodine and Rycal S107, which, like dantrolene, targets the ryanodine receptor, also promoted antisense-driven exon skipping, implicating the ryanodine receptor as the critical molecular target.

TCR engagement triggers the polarized recruitment of membrane, actin, and transducer assemblies within the T cell–APC contact that amplify and specify signaling cascades and Teffector activity. We report that caveolin-1, a scaffold that regulates polarity and signaling in nonlymphoid cells, is required for optimal TCR-induced actin polymerization, synaptic membrane raft polarity, and function in CD8, but not CD4, T cells. In CD8+ T cells, caveolin-1 ablation selectively impaired TCR-induced NFAT-dependent NFATc1 and cytokine gene expression, whereas caveolin-1 re-expression promoted NFATc1 gene expression. Alternatively, caveolin-1 ablation did not affect TCR-induced NF-κB–dependent Iκbα expression. Cav-1−/− mice did not efficiently promote CD8 immunity to lymphocytic choriomeningitis virus, nor did cav-1−/− OT-1+ CD8+ T cells efficiently respond to Listeria mono-cytogenes-OVA after transfer into wild-type hosts. Therefore, caveolin-1 is a T cell-intrinsic orchestrator of TCR-mediated membrane polarity and signal specificity selectively employed by CD8 T cells to customize TCR responsiveness.

The synthesis of N-Fmoc-O-(N'-Boc-N'-methyl)-aminohomoserine in 35% overall yield from l-homoserine is described. This amino acid can be efficiently incorporated into peptides using Fmoc-chemistry-based solid-phase peptide synthesis, and the resulting peptides can be chemoselectively glycosylated at the aminooxy side chains to generate neoglycopeptides. The synthesis of this derivative greatly expands the availability of a previously developed neoglycopeptide synthesis strategy.

BackgroundThe polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.Methodology/Principal FindingsHere we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.Conclusions/SignificanceThese findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.

Inadequate diagnostics compromise cancer care across lower- and middle-income countries (LMICs). We hypothesized that an inexpensive gene expression assay using paraffin-embedded biopsy specimens from LMICs could distinguish lymphoma subtypes without pathologist input. We reviewed all biopsy specimens obtained at the Instituto de Cancerología y Hospital Dr. Bernardo Del Valle in Guatemala City between 2006 and 2018 for suspicion of lymphoma. Diagnoses were established based on the World Health Organization classification and then binned into 9 categories: nonmalignant, aggressive B-cell, diffuse large B-cell, follicular, Hodgkin, mantle cell, marginal zone, natural killer/T-cell, or mature T-cell lymphoma. We established a chemical ligation probe-based assay (CLPA) that quantifies expression of 37 genes by capillary electrophoresis with reagent/consumable cost of approximately $10/sample. To assign bins based on gene expression, 13 models were evaluated as candidate base learners, and class probabilities from each model were then used as predictors in an extreme gradient boosting super learner. Cases with call probabilities < 60% were classified as indeterminate. Four (2%) of 194 biopsy specimens in storage <3 years experienced assay failure. Diagnostic samples were divided into 70% (n = 397) training and 30% (n = 163) validation cohorts. Overall accuracy for the validation cohort was 86% (95% confidence interval [CI]: 80%-91%). After excluding 28 (17%) indeterminate calls, accuracy increased to 94% (95% CI: 89%-97%). Concordance was 97% for a set of high-probability calls (n = 37) assayed by CLPA in both the United States and Guatemala. Accuracy for a cohort of relapsed/refractory biopsy specimens (n = 39) was 79% and 88%, respectively, after excluding indeterminate cases. Machine-learning analysis of gene expression accurately classifies paraffin-embedded lymphoma biopsy specimens and could transform diagnosis in LMICs.

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.