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. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC.

Type 1 immunity enables neonatal thymic ILC1 production.

Acute thymic atrophy occurs following type 1 inflammatory conditions such as viral infection and sepsis, resulting in cell death and disruption of T cell development. However, the impact type 1 immunity has on thymic-resident innate lymphoid cells (ILCs) remains unclear. Single-cell RNA sequencing revealed neonatal thymic-resident type 1 ILCs (ILC1s) as a unique and immature subset compared to ILC1s in other primary lymphoid organs. Culturing murine neonatal thymic lobes with the type 1 cytokines interleukin-12 (IL-12) and IL-18 resulted in a rapid expansion and thymic egress of KLRG1+CXCR6+ cytotoxic ILC1s. Live imaging showed the subcapsular thymic localization and exit of ILC1s following IL-12 + IL-18 stimulation. Similarly, murine cytomegalovirus infection in neonates resulted in thymic atrophy and subcapsular localization of thymic-resident ILC1s. Neonatal thymic grafting revealed that type 1 inflammation enhances the homing of cytokine-producing thymus-derived ILC1s to the liver and peritoneal cavity. Together, we show that type 1 immunity promotes the expansion and peripheral homing of thymic-derived ILC1s.

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. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

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

In the human thymus, a CD10+ PD-1+ TCRαß+ 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.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8ß expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

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.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

T-BET drives the conversion of human type 3 innate lymphoid cells into functional NK cells.

Type 3 innate lymphoid cells (ILC3s) are characterized by RORγt expression and they produce IL-22 upon activation. ILC3s play a role in maintenance of barrier integrity in the intestine. Under inflammatory conditions, the ILC composition of the mucosal tissues is altered due to a high degree of plasticity. It has been extensively demonstrated that both murine and human ILC3s convert into ILC1s to mediate appropriate immune responses. However, plasticity between human ILC3s and NK cells is less well documented. As T-BET and EOMES are key transcription factors in NK cell differentiation, we investigated whether ectopic T-BET or EOMES expression converts human ILC3s into NK cells. ILC3s with ectopic T-BET and EOMES expression downregulate RORγt expression, while T-BET-overexpressing ILC3s additionally upregulate EOMES expression. High E ctopic T-BET expression in ILC3s results in transdifferentiation towards CD94+ NK cells, whereas ectopic EOMES overexpression results in dedifferentiation of ILC3s into CD94-CD117-/low cells but is ineffective in NK cell generation. Dedifferentiating ILC3s from both T-BET and EOMES overexpression cultures upregulate NK cell receptors, perforin and granzyme B. Finally, IL-22 secretion is completely blocked in transdifferentiating ILC3s with both T-BET and EOMES ectopic expression, whereas only T-BET overexpression increases IFN-γ secretion and cytotoxicity. Altogether, these findings demonstrate that human ILC3s can convert into functional NK cells, wherein T-BET, and not EOMES, is the main driver.

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.

Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

Transcriptional dynamics and epigenetic regulation of E and ID protein encoding genes during human T cell development.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αß and γδ T cells. Although many of the mechanisms that control αß- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αß T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

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. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human haematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2Rß expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

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.

T-BET and EOMES Accelerate and Enhance Functional Differentiation of Human Natural Killer Cells.

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.

In vitro 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. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM-like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML.

A Novel Non-Coding Variant in DCLRE1C Results in Deregulated Splicing and Induces SCID Through the Generation of a Truncated ARTEMIS Protein That Fails to Support V(D)J Recombination and DNA Damage Repair.

Severe Combined Immune Deficiency (SCID) is a primary deficiency of the immune system in which opportunistic and recurring infections are often fatal during neonatal or infant life. SCID is caused by an increasing number of genetic defects that induce an abrogation of T lymphocyte development or function in which B and NK cells might be affected as well. Because of the increased availability and usage of next-generation sequencing (NGS), many novel variants in SCID genes are being identified and cause a heterogeneous disease spectrum. However, the molecular and functional implications of these new variants, of which some are non-coding, are often not characterized in detail. Using targeted NGS, we identified a novel homozygous c.465-1G>C splice acceptor site variant in the DCLRE1C gene in a T-B-NK+ SCID patient and fully characterized the molecular and functional impact. By performing a minigene splicing reporter assay, we revealed deregulated splicing of the DCLRE1C transcript since a cryptic splice acceptor in exon 7 was employed. This induced a frameshift and the generation of a p.Arg155Serfs*15 premature termination codon (PTC) within all DCLRE1C splice variants, resulting in the absence of full-length ARTEMIS protein. Consistently, a V(D)J recombination assay and a G0 micronucleus assay demonstrated the inability of the predicted mutant ARTEMIS protein to perform V(D)J recombination and DNA damage repair, respectively. Together, these experiments molecularly and functionally clarify how a newly identified c.465-1G>C variant in the DCLRE1C gene is responsible for inducing SCID. In a clinical context, this demonstrates how the experimental validation of new gene variants, that are identified by NGS, can facilitate the diagnosis of SCID which can be vital for implementing appropriate therapies.

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.

Treatment of a patient with severe cytomegalovirus (CMV) infection after haploidentical stem cell transplantation with donor derived CMV specific T cells.

Objectives: Cytomegalovirus (CMV) infection is one of the most common complications in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. The classic antiviral treatments have shown clinical efficacy but are often associated with drug resistance. Reconstitution of CMV-specific cellular immunity is essential in controlling CMV infection; therefore, adoptive transfer of CMV-specific T cells is a promising treatment option. We treated a patient with a multidrug resistant CMV infection after haploidentical HSCT with CMV-specific T cells.Methods: The T cells were derived from the HSCT donor who was CMV seropositive. We generated the T cells by a short-term Good Manufacturing Practice (GMP) grade protocol in which a leukapheresis product of the HSCT donor was stimulated with the immunodominant antigen pp65 and interferon-γ secreting cells were isolated. A total of 5 × 105 T cells were administered to the patient within 30 hours after leukapheresis.Results: The patient was closely monitored for reconstitution of antiviral T cell immunity and viral replication after adoptive T cell transfer. We observed an in vivo expansion of both CD4+ and CD8+ CMV-specific T cells associated with a significant decrease in viral burden and clinical improvement.Conclusion: This case report further supports the feasibility and effectiveness of adoptive donor T cell transfer for the treatment of drug resistant CMV infections after allo-HSCT.

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.

Conventional and Computational Flow Cytometry Analyses Reveal Sustained Human Intrathymic T Cell Development From Birth Until Puberty.

The thymus is the organ where subsets of mature T cells are generated which subsequently egress to function as central mediators in the immune system. While continuously generating T cells even into adulthood, the thymus does undergo involution during life. This is characterized by an initial rapid decrease in thymic cellularity during early life and by a second age-dependent decline in adulthood. The thymic cellularity of neonates remains low during the first month after birth and the tissue reaches a maximum in cellularity at 6 months of age. In order to study the effect that this first phase of thymic involution has on thymic immune subset frequencies, we performed multi-color flow cytometry on thymic samples collected from birth to 14 years of age. In consideration of the inherent limitations posed by conventional flow cytometry analysis, we established a novel computational analysis pipeline that is adapted from single-cell transcriptome sequencing data analysis. This allowed us to overcome technical effects by batch correction, analyze multiple samples simultaneously, limit computational cost by subsampling, and to rely on KNN-graphs for graph-based clustering. As a result, we successfully identified rare, distinct and gradually developing immune subsets within the human thymus tissues. Although the thymus undergoes early involution from infanthood onwards, our data suggests that this does not affect human T-cell development as we did not observe significant alterations in the proportions of T-lineage developmental intermediates from birth to puberty. Thus, in addition to providing an interesting novel strategy to analyze conventional flow cytometry data for the thymus, our work shows that the early phase of human thymic involution mainly limits the overall T cell output since no obvious changes in thymocyte subsets could be observed.

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.