ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase.

Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.

CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR).

Neoantigen-targeted dendritic cell vaccination in lung cancer patients induces long-lived T cells exhibiting the full differentiation spectrum.

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients.

HES6knockdown in human hematopoietic precursor cells reduces their in vivo engraftment potential and their capacity to differentiate into erythroid cells, B cells, T cells and plasmacytoid dendritic cells.

Hematopoiesis is driven by molecular mechanisms that induce differentiation and proliferation of hematopoietic stem cells and their progeny. This involves the activity of various transcription factors, such as members of the Hairy/Enhancer of Split (HES) family, and important roles for both HES1 and HES4 have been shown in normal and malignant hematopoiesis. Here, we investigated the role of HES6 in human hematopoiesis using in vitro and in vivo models.

The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation.

Single-cell profiling identifies a novel human polyclonal unconventional T cell lineage.

In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred.

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency.

Natural killer (NK) cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human.

Small-scale manufacturing of neoantigen-encoding messenger RNA for early-phase clinical trials.

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA.

OP9-DL1 co-culture and subsequent maturation in the presence of IL-21 generates tumor antigen-specific T cells with a favorable less-differentiated phenotype and enhanced functionality.

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms' tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g.

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells.

Human Thymic CD10 PD-1 Intraepithelial Lymphocyte Precursors Acquire Interleukin-15 Responsiveness at the CD1a CD95 CD28 CCR7 Developmental Stage.

Human thymic CD8αα CD10 PD-1 αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18).

T-cells with a single tumor antigen-specific T-cell receptor can be generated from clinically relevant stem cell sources.

Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens.

Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo.

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment.

Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls).

Nanobody Based Dual Specific CARs.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem.

The checkpoint for agonist selection precedes conventional selection in human thymus.

The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive.

Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach.