A metabolic dependency of EBV can be targeted to hinder B cell transformation.

Following infection of B cells, Epstein Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match ATP-production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

A method for polyclonal antigen-specific T cell-targeted genome editing (TarGET) for adoptive cell transfer applications.

Adoptive cell therapy of donor-derived, antigen-specific T cells expressing native T cell receptors (TCRs) is a powerful strategy to fight viral infections in immunocompromised patients. Determining the fate of T cells following patient infusion hinges on the ability to track them in vivo. While this is possible by genetic labeling of parent cells, the applicability of this approach has been limited by the non-specificity of the edited T cells. Here, we devised a method for CRISPR-targeted genome integration of a barcoded gene into Epstein-Barr virus-antigen-stimulated T cells and demonstrated its use for exclusively identifying expanded virus-specific cell lineages. Our method facilitated the enrichment of antigen-specific T cells, which then mediated improved cytotoxicity against Epstein-Barr virus-transformed target cells. Single-cell and deep sequencing for lineage tracing revealed the expansion profile of specific T cell clones and their corresponding gene expression signature. This approach has the potential to enhance the traceability and the monitoring capabilities during immunotherapeutic T cell regimens.

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing.

Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

Immunocompromised host section: Adoptive T-cell therapy for dsDNA viruses in allogeneic hematopoietic cell transplant recipients.

PURPOSE OF REVIEW: Double-stranded DNA (dsDNA) viruses remain important causes of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). As treatment options are limited, adoptive therapy with virus-specific T cells (VST) is promising in restoring immunity and thereby preventing and treating virus infections. Here we review current evidence and recent advances in the field of VST for dsDNA viruses in allogeneic HCT recipients. RECENT FINDINGS: Four different protocols for VST generation are currently used in clinical trials, and various products including multivirus-specific and off-the-shelf products are under investigation for prophylaxis, preemptive therapy or treatment. Data from nearly 1400 dsDNA-VST applications in allogeneic HCT patients have been published and demonstrated its safety. Although Epstein-Barr virus, cytomegalovirus, and adenovirus-specific T-cell therapy studies have predominated over the past 25 years, additional human herpes viruses were added to multivirus-specific T cells over the last decade and clinical evidence for polyomavirus-specific VST has just recently emerged. Response rates of around 70-80% have been reported, but cautious interpretation is warranted as data are predominantly from phase 1/2 studies and clinical efficacy needs to be confirmed in phase 3 studies. SUMMARY: Investigation on the 'ideal' composition of VST is ongoing. Several products recently entered phase 3 trials and may allow widespread clinical use in the near future.