Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF.

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

Distinct and temporary-restricted epigenetic mechanisms regulate human αß and γδ T cell development.

The development of TCRαß and TCRγδ T cells comprises a step-wise process in which regulatory events control differentiation and lineage outcome. To clarify these mechanisms, we employed RNA-sequencing, ATAC-sequencing and ChIPmentation on well-defined thymocyte subsets that represent the continuum of human T cell development. The chromatin accessibility dynamics show clear stage specificity and reveal that human T cell-lineage commitment is marked by GATA3- and BCL11B-dependent closing of PU.1 sites. A temporary increase in H3K27me3 without open chromatin modifications is unique for ß-selection, whereas emerging γδ T cells, which originate from common precursors of ß-selected cells, show large chromatin accessibility changes due to strong T cell receptor (TCR) signaling. Furthermore, we unravel distinct chromatin landscapes between CD4+ and CD8+ αß-lineage cells that support their effector functions and reveal gene-specific mechanisms that define mature T cells. This resource provides a framework for studying gene regulatory mechanisms that drive normal and malignant human T cell development.

Integrated scRNA-Seq Identifies Human Postnatal Thymus Seeding Progenitors and Regulatory Dynamics of Differentiating Immature Thymocytes.

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow-derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the nature of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both precursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics underlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signature of immature human thymocytes and provides a resource for the further study of human thymopoiesis.

HES6 knockdown 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. Using bulk and scRNA-seq data, we show that HES6 is expressed during erythroid/megakaryocyte and pDC development, as well as in multipotent precursors and at specific stages of T- and B-cell development following preBCR and preTCR signalling, respectively. Consistently, knockdown of HES6 in cord blood-derived hematopoietic precursors in well-defined in vitro differentiation assays resulted in reduced differentiation of human hematopoietic precursors towards megakaryocytes, erythrocytes, pDCs, Band T-cells. In addition, HES6 knockdown HSPCs displayed reduced colony forming unit capacity in vitro and impaired potential to reconstitute hematopoiesis in vivo in a competitive transplantation assay. We demonstrate that loss of HES6 expression impacts cell cycle progression during erythroid differentiation and provide evidence for potential downstream target genes that impact these perturbations. Thus, our study uncovers new insights for a role of HES6 in human hematopoiesis.

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation.

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and they regulate other immune cells by cytokine secretion. Although murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells and by expressing the dominant-negative ETS1 p27 isoform in cord blood hematopoietic progenitor cells, we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation (ie, E4BP4, TXNIP, TBET, GATA3, HOBIT, BLIMP1). In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation.

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. The authors have developed MIDRIXNEO, a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIXNEO. To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described.

Distinct Notch1 and BCL11B requirements mediate human γδ/αß T cell development.

γδ and αß T cells have unique roles in immunity and both originate in the thymus from T-lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αß-lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR-17-92 cluster is a Notch1 target in immature thymocytes and that miR-17 can restrict BCL11B expression in these Notch-dependent T cell precursors. We show that enforced miR-17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αß but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage-specific Notch-driven negative feedback loop through which miR-17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease-associated genes BCL11B and the miR-17-92 cluster in a human context.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

HES1 and HES4 have non-redundant roles downstream of Notch during early human T-cell development.

In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34+ hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is not capable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is not sufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions.

TCR Sequencing Reveals the Distinct Development of Fetal and Adult Human Vγ9Vδ2 T Cells.

Phosphoantigen-reactive Vγ9Vδ2 T cells represent the main innate human γδ T cell subset and dominate the fetal and adult peripheral blood γδ T cell repertoire. It has been hypothesized that adult blood Vγ9Vδ2 T cells find their origin in the fetus like it is established for mouse innate γδ T cells. To address this issue, we analyzed the CDR3 of the TCR of human blood and thymic Vγ9Vδ2 T cells from fetal until adult life. We first identified key differences in the CDR3 repertoire of fetal and adult blood Vγ9Vδ2 T cells, including in CDR3 features important for phosphoantigen reactivity. Next, we showed that most of these key adult CDR3 features were already present in the postnatal thymus and were further enhanced upon selection in vitro by the microbial-derived phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. Finally, we demonstrated that the generation of a fetal-type or adult-type Vγ9Vδ2 CDR3 repertoire is determined by the fetal and postnatal nature of the hematopoietic stem and precursor cell. Thus, our data indicate that fetal blood Vγ9Vδ2 T cells find their origin in the fetal thymus whereas adult blood Vγ9Vδ2 T cells are generated to a large degree independently after birth.

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 de novo 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). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

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+ αß 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). We showed that only the CD1a- fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10+ PD-1+ population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a- CD10+ PD-1+ population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.

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. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.

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. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity.

The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression.

Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: ApcMin/+-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer.

Notch3 activation is sufficient but not required for inducing human T-lineage specification.

Although the role for the individual Notch receptors in early hematopoiesis have been thoroughly investigated in mouse, studies in human have been mostly limited to the use of pan-Notch inhibitors. However, such studies in human are important to predict potential side effects of specific Notch receptor blocking reagents because these are currently being considered as therapeutic tools to treat various Notch-dependent diseases. In this study, we studied the individual roles of Notch1 and Notch3 in early human hematopoietic lineage decisions, particularly during T-lineage specification. Although this process in mice is solely dependent on Notch1 activation, we recently reported Notch3 expression in human uncommitted thymocytes, raising the possibility that Notch3 mediates human T-lineage specification. Although expression of a constitutive activated form of Notch3 (ICN3) results in the induction of T-lineage specification in human CD34(+) hematopoietic progenitor cells, similar to ICN1 overexpression, loss-of-function studies using blocking Abs reveal that only Notch1, but not Notch3, is critical in this process. Blocking of Notch1 activation in OP9-DLL4 cocultures resulted in a complete block in T-lineage specification and induced monocytic and plasmacytoid dendritic cell differentiation instead. In fetal thymus organ cultures, impeded Notch1 activation resulted in B and dendritic cell development. In contrast, Notch3 blocking Abs only marginally affected T-lineage specification and hematopoietic differentiation with a slight increase in monocyte development. No induction of B or dendritic cell development was observed. Thus, our results unambiguously reveal a nonredundant role for Notch1 in human T-lineage specification, despite the expression of other Notch receptors.

In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis.

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.

Differential Ly49e expression pathways in resting versus TCR-activated intraepithelial γδ T cells.

The Ly49 NK receptor family in mice is composed of several members that recognize MHC class I (MHC-I) or MHC-I-related molecules. We and others have shown before that Ly49E is a unique member, with a different expression pattern on NK cells and being triggered by the non-MHC-I-related protein urokinase plasminogen activator. Among the entire Ly49 receptor family, Ly49E is the only Ly49 member expressed by epidermal-localized γδ T cells and their fetal thymic TCRγδ precursors, and it is the most abundantly expressed member on intestinal intraepithelial γδ T cell lymphocytes. In this study, we provide mechanistic insights into the regulation of Ly49e expression in γδ T cells. First, we demonstrate that TCR-mediated activation of intraepithelial γδ T cells significantly increases Ly49E expression. This results from de novo Ly49E expression and is highly selective, because no other Ly49 family members are induced. TCR-mediated Ly49E induction is a conserved feature of skin- and gut-residing intraepithelial-localized γδ T cell subsets, whereas it is not observed in spleen γδ T cells. By investigating Ly49e promoter activities and lymphotoxin (LT) αß dependency in resting versus TCR-activated intraepithelial γδ T cells, we reveal two separate regulatory pathways for Ly49E expression, as follows: a LTαß-dependent pathway leading to basal Ly49E expression in resting cells that is induced by Pro2-mediated Ly49e transcription, and a LTαß-independent pathway leading to elevated, Pro3-driven Ly49E expression in TCR-stimulated cells.

Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated, site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study, we demonstrate the ability to target the integration of WAS2-12-containing constructs into intron 1 of the endogenous WAS gene of primary CD34+ hematopoietic stem and progenitor cells (HSPCs), as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore, TI restored WASp-dependent function to WAS patient T cells. Edited CD34+ HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient's CD34+ cells.

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). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy, rather CD70 interaction in cis with the nanoCAR induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9, resulted in dramatically enhanced functionality in the DLBCL PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knock out (KO) CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that WT CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. On the other hand, the gene signature of KO CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products that led to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.