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We performed a Phase I clinical trial of donor derived CD19-specific chimeric antigen receptor T-cells (CAR T-cells) for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene capacity limitations of traditional viral vectors, CAR T-cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, one patient developed a gradually enlarging retroperitoneal tumor due to a CAR expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection of a second CAR T-cell tumor in thoracic para-aortic lymph nodes in an asymptomatic patient. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell derived lymphoma progressed and one patient died. We describe the first two cases of malignant lymphoma derived from CAR gene modified T-cells. Although CAR T-cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. The trial was registered at www.anzctr.org.au as ACTRN12617001579381.

No abstract

The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. Cell-to-cell variability of BCR sequences due to V(D)J recombination and somatic hypermutation (SHM) necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing (scRNA-seq) presents the opportunity for simultaneous capture of the BCR sequence and transcriptomic signature for a detailed understanding of the dynamics of an immune response.We developed VDJPuzzle 2.0, a bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains. VDJPuzzle successfully reconstructs BCRs from 98.3% (n=117) of human and 96.5% (n=200) from murine B cells. 92.0% of clonotypes and 90.3% of mutations were concordant with single-cell Sanger sequencing of the immunoglobulin chains.

Supplementary data are available at Bioinformatics online.

In recent years, proteomic data have contributed to genome annotation efforts, most notably in humans and mice, and spawned a field termed "proteogenomics". Yeast, in contrast with higher eukaryotes, has a small genome, which has lent itself to simpler ORF prediction. Despite this, continual advances in mass spectrometry suggest that proteomics should be able to improve genome annotation even in this well-characterized species. Here we applied a proteogenomics workflow to yeast to identify novel protein-coding genes. Specific databases were generated, from intergenic regions of the genome, which were then queried with MS/MS data. This suggested the existence of several putative novel ORFs of <100 codons, one of which we chose to validate. Synthetic peptides, RNA-Seq analysis, and evidence of evolutionary conservation allowed for the unequivocal definition of a new protein of 78 amino acids encoded on chromosome X, which we dub YJR107C-A. It encodes a new type of domain, which ab initio modeling suggests as predominantly α-helical. We show that this gene is nonessential for growth; however, deletion increases sensitivity to osmotic stress. Finally, from the above discovery process, we discuss a generalizable strategy for the identification of short ORFs and small proteins, many of which are likely to be undiscovered.

The emergence of the SARS-CoV-2 variant-of-concern Omicron (B.1.1.529) has destabilized global efforts to control the impact of COVID-19. Recent data have suggested that B.1.1.529 can readily infect people with naturally acquired or vaccine-induced immunity, facilitated in some cases by viral escape from antibodies that neutralize ancestral SARS-CoV-2. However, severe disease appears to be relatively uncommon in such individuals, highlighting a potential role for other components of the adaptive immune system. We report here that SARS-CoV-2 spike-specific CD4+ and CD8+ T cells induced by prior infection and, more extensively, by mRNA vaccination provide comprehensive heterologous immune reactivity against B.1.1.529. Pairwise comparisons across groups further revealed that SARS-CoV-2 spike-reactive CD4+ and CD8+ T cells exhibited similar functional attributes, memory distributions, and phenotypic traits in response to the ancestral strain or B.1.1.529. Our data indicate that established SARS-CoV-2 spike-specific CD4+ and CD8+ T cell responses, especially after mRNA vaccination, remain largely intact against B.1.1.529.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature Medicine is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.