An Aggregation-Induced Emission-Based Dual Emitting Nanoprobe for Detecting Intracellular pH and Unravelling Metabolic Variations in Differentiating Lymphocytes.

Monitoring T lymphocyte differentiation is essential for understanding T cell fate regulation and advancing adoptive T cell immunotherapy. However, current biomarker analysis methods necessitate cell lysis, leading to source depletion. Intracellular pH (pHi) can be affected by the presence of lactic acid (LA), a metabolic mediator of T cell activity such as glycolysis during T cell activation; therefore, it is a potentially a good biomarker of T cell state. In this work, a dual emitting enhancement-based nanoprobe, namely, AIEgen@F127-AptCD8, was developed to accurately detect the pHi of T cells to "read" the T cell differentiation process. The nanocore of this probe comprises a pair of AIE dyes, TPE-AMC (pH-sensitive moiety) and TPE-TCF, that form a donor-acceptor pair for sensitive detection of pHi by dual emitting enhancement analysis. The nanoprobe exhibits a distinctly sensitive narrow range of pHi values (from 6.0 to 7.4) that can precisely distinguish the differentiated lymphocytes from naïve ones based on their distinct pHi profiles. Activated CD8+ T cells demonstrate lower pHi (6.49 ± 0.09) than the naïve cells (7.26 ± 0.11); Jurkat cells exhibit lower pHi (6.43 ± 0.06) compared to that of nonactivated ones (7.29 ± 0.09) on 7 days post-activation. The glycolytic product profiles in T cells strongly correlate with their pHi profiles, ascertaining the reliability of probing pHi for predicting T cell states. The specificity and dynamic detection capabilities of this nanoprobe make it a promising tool for indirectly and noninvasively monitoring T cell activation and differentiation states.

Transient PU.1 low fetal progenitors generate lymphoid progeny that contribute to adult immunity.

Hematopoietic stem cells and multipotential progenitors emerge in multiple, overlapping waves of fetal development. Some of these populations seed the bone marrow and sustain adult B- and T-cell development long-term after birth. However, others are present transiently, but whether they are vestigial or generate B and T cells that contribute to the adult immune system is not well understood. We now report that transient fetal progenitors distinguished by expression of low levels of the PU.1 transcription factor generated activated and memory T and B cells that colonized and were maintained in secondary lymphoid tissues. These included the small and large intestines, where they may contribute to the maintenance of gut homeostasis through at least middle age. At least some of the activated/memory cells may have been the progeny of B-1 and marginal zone B cells, as transient PU.1low fetal progenitors efficiently generated those populations. Taken together, our data demonstrate the potential of B- and T-cell progeny of transient PU.1low fetal progenitors to make an early and long-term contribution to the adult immune system.

DNA Reaction Circuits to Establish Designated Biological Functions in Multicellular Community.

In multicellular organisms, individual cells are coordinated through complex communication networks to accomplish various physiological tasks. Aiming to establish new biological functions in the multicellular community, we used DNA as the building block to develop a cascade of nongenetic reaction circuits to establish a dynamic cell-cell communication network. Utilizing membrane-anchored amphiphilic DNA tetrahedra (TDN) as the nanoscaffold, reaction circuits were incorporated into three unrelated cells in order to uniquely regulate their sense-and-response behaviors. As a proof-of-concept, this step enabled these cells to simulate significant biological events involved in T cell-mediated anticancer immunity. Such events included cancer-associated antigen recognition and the presentation of antigen-presenting cells (APCs), APC-facilitated T cell activation and dissociation, and T cell-mediated cancer targeting and killing. By combining the excellent programmability and molecular recognition ability of DNA, our cell-surface reaction circuits hold promise for mimicking and manipulating many biological processes.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering.

ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient's own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).

A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Structures of human γδ T cell receptor-CD3 complex.

Gamma delta (γδ) T cells, a unique T cell subgroup, are crucial in various immune responses and immunopathology1-3. The γδ T cell receptor (TCR), which is generated by γδ T cells, recognizes a diverse range of antigens independently of the major histocompatibility complex2. The γδ TCR associates with CD3 subunits, initiating T cell activation and holding great potential in immunotherapy4. Here we report the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR-CD3 complexes5,6, revealing two distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR-CD3 complex is monomeric, with considerable conformational flexibility in the TCRγ-TCRδ extracellular domain and connecting peptides. The length of the connecting peptides regulates the ligand association and T cell activation. A cholesterol-like molecule wedges into the transmembrane region, exerting an inhibitory role in TCR signalling. The Vγ5Vδ1 TCR-CD3 complex displays a dimeric architecture, whereby two protomers nestle back to back through the Vγ5 domains of the TCR extracellular domains. Our biochemical and biophysical assays further corroborate the dimeric structure. Importantly, the dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. These findings reveal organizing principles of the γδ TCR-CD3 complex, providing insights into the unique properties of γδ TCR and facilitating immunotherapeutic interventions.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

A Scalable Microfluidic Platform for Nanoparticle Formulation: For Exploratory- and Industrial-Level Scales.

Nanoparticle synthesis on microfluidic platforms provides excellent reproducibility and control over bulk synthesis. While there have been plenty of platforms for producing nanoparticles (NPs) with controlled physicochemical properties, such platforms often operate in a narrow range of predefined flow rates. The flow rate limitation restricts either up-scalability for industrial production or down-scalability for exploratory research use. Here, we present a universal flow rate platform that operates over a wide range of flow rates (0.1-75 mL/min) for small-scale exploratory research and industrial-level synthesis of NPs without compromising the mixing capabilities. The wide range of flow rate is obtained by using a coaxial flow with a triangular microstructure to create a vortex regardless of the flow regime (Reynolds number). The chip synthesizes several types of NPs for gene and protein delivery, including polyplex, lipid NPs, and solid polymer NPs via self-assembly and precipitation, and successfully expresses GFP plasmid DNA in human T cells.

Mis-expression of GATA6 re-programs cell fate during early hematopoiesis.

The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

FOXO1 is a master regulator of memory programming in CAR T cells.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics.

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows 'on-the-fly' sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 µm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

Spatially resolved quantification of oxygen consumption rate in ex vivo lymph node slices.

Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in ex vivo tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.

Cryopreserved leukapheresis material can be transferred from controlled rate freezers to ultracold storage at warmer temperatures without affecting downstream CAR-T cell culture performance and in-vitro functionality.

Chimeric antigen receptor (CAR) T-cell therapies are increasingly adopted as a commercially available treatment for hematologic and solid tumor cancers. As CAR-T therapies reach more patients globally, the cryopreservation and banking of patients' leukapheresis materials is becoming imperative to accommodate intra/inter-national shipping logistical delays and provide greater manufacturing flexibility. This study aims to determine the optimal temperature range for transferring cryopreserved leukapheresis materials from two distinct types of controlled rate freezing systems, Liquid Nitrogen (LN2)-based and LN2-free Conduction Cooling-based, to the ultracold LN2 storage freezer (≤-135 °C), and its impact on CAR T-cell production and functionality. Presented findings demonstrate that there is no significant influence on CAR T-cell expansion, differentiation, or downstream in-vitro function when employing a transfer temperature range spanning from -30 °C to -80 °C for the LN2-based controlled rate freezers as well as for conduction cooling controlled rate freezers. Notably, CAR T-cells generated from cryopreserved leukapheresis materials using the conduction cooling controlled rate freezer exhibited suboptimal performance in certain donors at transfer temperatures lower than -60 °C, possibly due to the reduced cooling rate of lower than 1 °C/min and extended dwelling time needed to reach the final temperatures within these systems. This cohort of data suggests that there is a low risk to transfer cryopreserved leukapheresis materials at higher temperatures (between -30 °C and -60 °C) with good functional recovery using either controlled cooling system, and the cryopreserved materials are suitable to use as the starting material for autologous CAR T-cell therapies.

Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

Lyophilized lymph nodes for improved delivery of chimeric antigen receptor T cells.

Lymph nodes are crucial organs of the adaptive immune system, orchestrating T cell priming, activation and tolerance. T cell activity and function are highly regulated by lymph nodes, which have a unique structure harbouring distinct cells that work together to detect and respond to pathogen-derived antigens. Here we show that implanted patient-derived freeze-dried lymph nodes loaded with chimeric antigen receptor T cells improve delivery to solid tumours and inhibit tumour recurrence after surgery. Chimeric antigen receptor T cells can be effectively loaded into lyophilized lymph nodes, whose unaltered meshwork and cytokine and chemokine contents promote chimeric antigen receptor T cell viability and activation. In mouse models of cell-line-derived human cervical cancer and patient-derived pancreatic cancer, delivery of chimeric antigen receptor T cells targeting mesothelin via the freeze-dried lymph nodes is more effective in preventing tumour recurrence when compared to hydrogels containing T-cell-supporting cytokines. This tissue-mediated cell delivery strategy holds promise for controlled release of various cells and therapeutics with long-term activity and augmented function.

AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.