Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

The microRNA cluster miR-17∼92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.

Follicular helper T cells (TFH cells) are the prototypic helper T cell subset specialized to enable B cells to form germinal centers (GCs) and produce high-affinity antibodies. We found that expression of microRNAs (miRNAs) by T cells was essential for TFH cell differentiation. More specifically, we show that after immunization of mice with protein, the miRNA cluster miR-17∼92 was critical for robust differentiation and function of TFH cells in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17∼92 restrained the expression of genes 'inappropriate' to the TFH cell subset, including the direct miR-17∼92 target Rora. Removal of one Rora allele partially 'rescued' the inappropriate gene signature in miR-17∼92-deficient TFH cells. Our results identify the miR-17∼92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene-expression program.

MicroRNAs 24 and 27 Suppress Allergic Inflammation and Target a Network of Regulators of T Helper 2 Cell-Associated Cytokine Production.

MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.

Colony environment and absence of brood enhance tolerance to a neonicotinoid in winter honey bee workers, Apis mellifera.

In eusocial insects, worker longevity is essential to ensure colony survival in brood-free periods. Trade-offs between longevity and other traits may render long-living workers in brood-free periods more susceptible to pesticides compared to short-lived ones. Further, colony environment (e.g., adequate nutrition) may enable workers to better cope with pesticides, yet data comparing long vs. short-living workers and the role of the colony environment for pesticide tolerance are scarce. Here, we show that long-living honey bee workers, Apis mellifera, are less susceptible to the neonicotinoid thiamethoxam than short-lived workers, and that susceptibility was further reduced when workers were acclimatized under colony compared to laboratory conditions. Following an OECD protocol, freshly-emerged workers were exposed to thiamethoxam in summer and winter and either acclimatized within their colony or in the laboratory. Mortality and sucrose consumption were measured daily and revealed that winter workers were significantly less susceptible than summer workers, despite being exposed to higher thiamethoxam dosages due to increased food consumption. Disparencies in fat body activity, which is key for detoxification, may explain why winter bees were less susceptible. Furthermore, colony acclimatization significantly reduced susceptibility towards thiamethoxam in winter workers likely due to enhanced protein nutrition. Brood absence and colony environment seem to govern workers' ability to cope with pesticides, which should be considered in risk assessments. Since honey bee colony losses occur mostly over winter, long-term studies assessing the effects of pesticide exposure on winter bees are required to better understand the underlying mechanisms.

Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo.

Regulatory T cells (T(reg) cells) are central to the maintenance of immune homeostasis. However, little is known about the stability of T(reg) cells in vivo. In this study, we demonstrate that a substantial percentage of cells had transient or unstable expression of the transcription factor Foxp3. These 'exFoxp3' T cells had an activated-memory T cell phenotype and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers were higher in inflamed tissues in autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid onset of diabetes. Finally, analysis of the T cell receptor repertoire suggested that exFoxp3 cells developed from both natural and adaptive T(reg) cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells.

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

Under Review: Data Requirements and Method Development of a New Bee Risk Assessment Scheme for Plant Protection Product Registration.

An up-to-date ecotoxicological risk assessment of plant protection products (PPPs) depends on the constant improvement of risk assessment methods and guidelines, and a thorough evaluation of their impacts. Here, we explain how the risk assessment of PPPs with regard to bees and the authorisation of PPPs is conducted in Switzerland. We further report the design and application of a new method to study homing flights of honey bees using the Radio Frequency Identification (RFID) technique. The new method allowed to address the effects of sublethal doses of two neonicotinoids, thiamethoxam and thiacloprid, on the flight capacities of honey bees. Currently, this study design is under evaluation in an international ring test, in which the Swiss Bee Research centre participates. It is the first test design focussing on sublethal effects of PPPs on honey bees and a draft method will be submitted to OECD to become an official test guideline in the near future. Potential shortcomings and ideas for refinements on the RFID test design are discussed.

MicroRNA regulation of T-cell differentiation and function.

MicroRNAs (miRNAs) are emerging as key controllers of T-cell differentiation and function. Their expression is dynamically regulated by extracellular signals such as costimulation and cytokine signals. miRNAs set thresholds for gene expression and optimize protein concentrations of genetic networks. Absence of individual miRNAs can lead to severe immune dysfunction. In this study, we review emerging principles and provide examples of important functions exerted by miRNAs. Although our understanding of miRNA function in T-cell differentiation is still rudimentary, the available evidence leaves no doubt that these small post-transcriptional regulators are indispensable for proper functioning of the immune system.

A subpopulation of CD103(pos) ICOS(pos) Treg cells occurs at high frequency in lymphopenic mice and represents a lymph node specific differentiation stage.

Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self-reactive, chronic, and homeostatic T-cell responses. Here, we report that the increase in Treg-cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103(pos) ICOS(pos) Treg cells. Though present in the thymus, CD103(pos) ICOS(pos) Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103(pos) ICOS(pos) Treg cells is not affected by the absence of IL-7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103(pos) ICOS(pos) Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103(pos) ICOS(pos) phenotype represents a LN-specific differentiation stage arrived at by several other Treg-cell subsets. Thus, tissue-specific cues determine the overall potency of the peripheral Treg-cell pool by shaping its subset composition.

Canonical microRNAs in thymic epithelial cells promote central tolerance.

Medullary thymic epithelial cells (mTECs) facilitate the deletion of developing self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens, a process which largely depends on the expression of the autoimmune regulator (Aire) gene. Mature microRNAs (miRNAs) that regulate gene expression post-transcriptionally are generated in a multistep process. The microprocessor complex, including DGCR8, cleaves canonical miRNAs, but alternative DGCR8-independent miRNA biogenesis pathways exist as well. In order to study the role of canonical miRNAs in thymic epithelial cells (TECs), we ablated Dgcr8 using a FoxN1-Cre transgene. We report that DGCR8-deficient TECs are unable to maintain proper thymic architecture and exhibit a dramatic loss of thymic cellularity. Importantly, DGCR8-deficient TECs develop a severe loss of Aire(+) mTECs. Using a novel immunization approach to amplify and detect self-reactive T cells within a polyclonal TCR repertoire, we demonstrate a link between the loss of Aire expression in DGCR8-deficient TECs and the breakdown of negative selection in the thymus. Thus, DGCR8 and canonical miRNAs are important in TECs for supporting central tolerance.

microRNA-17-92 regulates IL-10 production by regulatory T cells and control of experimental autoimmune encephalomyelitis.

microRNAs (miRNA) are essential for regulatory T cell (Treg) function but little is known about the functional relevance of individual miRNA loci. We identified the miR-17-92 cluster as CD28 costimulation dependent, suggesting that it may be key for Treg development and function. Although overall immune homeostasis was maintained in mice with miR-17-92-deficient Tregs, expression of the miR-17-92 miRNA cluster was critical for Treg accumulation and function during an acute organ-specific autoimmune disease in vivo. Treg-specific loss of miR-17-92 expression resulted in exacerbated experimental autoimmune encephalitis and failure to establish clinical remission. Using peptide-MHC tetramers, we demonstrate that the miR-17-92 cluster was specifically required for the accumulation of activated Ag-specific Treg and for differentiation into IL-10-producing effector Treg.

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation.

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.

Novel fungicide and neonicotinoid insecticide impair flight behavior in pollen foraging honey bees, Apis mellifera.

Bees are often exposed to pesticides affecting physiological functions and molecular mechanisms. Studies showed a potential link between altered expression of energy metabolism related transcripts and increased homing flight time of foragers exposed to pesticides. In this study, we investigated the effects of thiamethoxam and pyraclostrobin on longevity, flight behavior, and expression of transcripts involved in endocrine regulation (hbg-3, buffy, vitellogenin) and energy metabolism (cox5a, cox5b, cox17) using radio frequency identification (RFID) technology and quantitative polymerase chain reaction. Parallel, a laboratory study was conducted investigating whether pesticide exposure alone without the influence of flight activity caused similar expression patterns as in the RFID experiment. No significant effect on survival, homing flight duration, or return rate of exposed bees was detected. The overall time foragers spent outside the hive was significantly reduced post-exposure. Irrespective of the treatment group, a correlation was observed between cox5a, cox5b, cox17 and hbg-3 expression and prolonged homing flight duration. Our results suggest that flight behavior can impact gene expression and exposure to pesticides adversely affects the expression of genes that are important for maintaining optimal flight capacity. Our laboratory-based experiment showed significantly altered expression levels of cox5a, cox6c, and cox17. However, further work is needed to identify transcriptional profiles responsible for prolonged homing flight duration.

Insecticide exposure alters flight-dependent gene-expression in honey bees, Apis mellifera.

The increased reports of wild bee declines and annual losses of managed bees pose a significant threat to biodiversity and agricultural productivity. While these losses and declines are likely driven by various factors, the exposure of bees to agrochemicals has raised significant concern due to their ubiquitous use and potential adverse effects. Despite numerous studies suggesting neonicotinoids can negatively affect bees at the behavioral and molecular level, data linking these two factors remains sparse. Here we provide data on the impact of an acute dose of the neonicotinoid thiamethoxam on the flight performance and molecular transcription profiles of foraging honey bees (Apis mellifera). Using a controlled experimental design with tethered flight mills, we measured flight distance, duration, and speed, alongside the expression of genes involved in energy metabolism, hormone regulation, and biosynthesis. Acute thiamethoxam exposure resulted in hyperactive flight behavior but led to significant dysregulation of genes associated with oxidative phosphorylation, indicating potential disruptions in cellular energy production. These molecular changes were particularly evident when bees engaged in flight activities, suggesting that the combined stress of pesticide exposure and physical exertion exacerbates negative outcomes. Our study provides new insights into the molecular mechanisms underlying neonicotinoid-induced impairments in bee physiology that can help understand the potential long-term consequences of xenobiotic exposure on the foraging abilities of bees and ultimately fitness.

Chronic oral toxicity protocol for adult solitary bees (Osmia bicornis L.): Reduced survival under long-term exposure to a "bee-safe" insecticide.

Pollinators are essential for crop productivity. Yet, in agricultural areas, they may be threatened by pesticide exposure. Current pesticide risk assessments predominantly focus on honey bees, with a lack of standardized protocols for solitary bees. This study addresses this gap by developing a long-term oral exposure protocol tailored for O. bicornis. We conducted initial trials to determine optimal container sizes and feeding methods, ensuring high survival rates and accurate syrup consumption measurements. A validation test involving five laboratories was then conducted with the insecticide Flupyradifurone (FPF). Control mortality thresholds were set at ≤ 15% at 10 days. Three laboratories achieved ≤10%, demonstrating the protocol's effectiveness in maintaining healthy test populations. The seasonal timing of experiments influenced control mortality, underscoring the importance of aligning tests with the natural flight period of the population used. Our findings revealed dose-dependent effects of FPF on syrup consumption, showing stimulatory effects at lower concentrations and inhibitory effects at higher ones. The 10-day median lethal daily dose (LDD50) of FPF for O. bicornis (531.92 ng/bee/day) was 3.4-fold lower than that reported for Apis mellifera (1830 ng/bee/day), indicating Osmia's higher susceptibility. Unlike other insecticides, FPF did not exhibit time-reinforced toxicity. This study introduces a robust protocol for chronic pesticide exposure in solitary bees, addressing a critical gap in current risk assessment. Based on its low risk to honey bees and bumblebees, FPF is approved for application during flowering. However, our results suggest that it may threaten Osmia populations under realistic field conditions. Our findings underscore the need for comparative toxicity studies to ensure comprehensive protection of all pollinators and the importance of accounting for long term exposure scenarios in risk assessment. By enhancing our understanding of chronic pesticide effects in solitary bees, our study should contribute to the development of more effective conservation strategies and sustainable agricultural practices.

Melanoma Clonal Heterogeneity Leads to Secondary Resistance after Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is effective in patients with melanoma, although long-term responses seem restricted in patients who have complete remissions. Many patients develop secondary resistance to TIL-ACT but the involved mechanisms are unclear. In this study, we describe a case of secondary resistance to TIL-ACT possibly due to intratumoral heterogeneity and selection of a resistant tumor cell clone by the transferred T cells. To the best our knowledge, this is the first case of clonal selection of a pre-existing nondominant tumor cell clone; this report demonstrates the mechanism involved in secondary resistance to TIL-ACT that can potentially change current clinical practice because it advocates for T-cell collection from multiple tumor sites and analysis of tumor heterogeneity before treatment with TIL-ACT.

TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

Leveraging microRNAs for cellular therapy.

Owing to Karl Landsteiner's discovery of blood groups, blood transfusions became safe cellular therapies in the early 1900s. Since then, cellular therapy made great advances from transfusions with unmodified cells to today's commercially available chimeric antigen receptor (CAR) T cells requiring complex manufacturing. Modern cellular therapy products can be improved using basic knowledge of cell biology and molecular genetics. Emerging genome engineering tools are becoming ever more versatile and precise and thus catalyze rapid progress towards programmable therapeutic cells that compute input and respond with defined output. Despite a large body of literature describing important functions of non-coding RNAs including microRNAs (miRNAs), the vast majority of cell engineering efforts focuses on proteins. However, miRNAs form an important layer of posttranscriptional regulation of gene expression. Here, we highlight examples of how miRNAs can successfully be incorporated into engineered cellular therapies.