Fate Mapping and Quantitation of Hematopoiesis In Vivo.

Hematopoietic stem cells (HSCs) and downstream progenitors have long been studied based on phenotype, cell purification, proliferation, and transplantation into myeloablated recipients. These experiments, complemented by data on expression profiles, mouse mutants, and humans with hematopoietic defects, are the foundation for the current hematopoietic differentiation tree. However, there are fundamental gaps in our knowledge of the quantitative and qualitative operation of the HSC/progenitor system under physiological and pathological conditions in vivo. The hallmarks of HSCs, self-renewal and multipotency, are observed in in vitro assays and cell transplantation experiments; however, the extent to which these features occur naturally in HSCs and progenitors remains uncertain. We focus here on work that strives to address these unresolved questions, with emphasis on fate mapping and modeling of the hematopoietic flow from stem cells toward myeloid and lymphoid lineages during development and adult life.

A type 1 immunity-restricted promoter of the IL-33 receptor gene directs antiviral T-cell responses.

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33-ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33-ST2 axis in infections and inflammatory diseases.

Pyrimidine de novo synthesis inhibition selectively blocks effector but not memory T cell development.

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

Early emergence of T central memory precursors programs clonal dominance during chronic viral infection.

Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called 'memory inflation' is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family's capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family's long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.

Graft-versus-host disease is locally maintained in target tissues by resident progenitor-like T cells.

In allogeneic hematopoietic stem cell transplantation, donor αß T cells attack recipient tissues, causing graft-versus-host disease (GVHD), a major cause of morbidity and mortality. A central question has been how GVHD is sustained despite T cell exhaustion from chronic antigen stimulation. The current model for GVHD holds that disease is maintained through the continued recruitment of alloreactive effectors from blood into affected tissues. Here, we show, using multiple approaches including parabiosis of mice with GVHD, that GVHD is instead primarily maintained locally within diseased tissues. By tracking 1,203 alloreactive T cell clones, we fitted a mathematical model predicting that within each tissue a small number of progenitor T cells maintain a larger effector pool. Consistent with this, we identified a tissue-resident TCF-1+ subpopulation that preferentially engrafted, expanded, and differentiated into effectors upon adoptive transfer. These results suggest that therapies targeting affected tissues and progenitor T cells within them would be effective.

Fate mapping of single NK cells identifies a type 1 innate lymphoid-like lineage that bridges innate and adaptive recognition of viral infection.

Upon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.

Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Hypoxia is a common feature of solid tumors, and cells adapt by upregulating the transcription factor HIF-1α. Here, we defined the transcriptional landscape of mouse tumor-infiltrating natural killer (NK) cells by using single-cell RNA sequencing. Conditional deletion of Hif1a in NK cells resulted in reduced tumor growth, elevated expression of activation markers, effector molecules, and an enriched NF-κB pathway in tumor-infiltrating NK cells. Interleukin-18 (IL-18) from myeloid cells was required for NF-κB activation and the enhanced anti-tumor activity of Hif1a-/- NK cells. Extended culture with an HIF-1α inhibitor increased human NK cell responses. Low HIF1A expression was associated with high expression of IFNG in human tumor-infiltrating NK cells, and an enriched NK-IL18-IFNG signature in solid tumors correlated with increased overall patient survival. Thus, inhibition of HIF-1α unleashes NK cell anti-tumor activity and could be exploited for cancer therapy.

Single-Cell Tracing Dissects Regulation of Maintenance and Inheritance of Transcriptional Reinduction Memory.

Transcriptional memory of gene expression enables adaptation to repeated stimuli across many organisms. However, the regulation and heritability of transcriptional memory in single cells and through divisions remains poorly understood. Here, we combined microfluidics with single-cell live imaging to monitor Saccharomyces cerevisiae galactokinase 1 (GAL1) expression over multiple generations. By applying pedigree analysis, we dissected and quantified the maintenance and inheritance of transcriptional reinduction memory in individual cells through multiple divisions. We systematically screened for loss- and gain-of-memory knockouts to identify memory regulators in thousands of single cells. We identified new loss-of-memory mutants, which affect memory inheritance into progeny. We also unveiled a gain-of-memory mutant, elp6Δ, and suggest that this new phenotype can be mediated through decreased histone occupancy at the GAL1 promoter. Our work uncovers principles of maintenance and inheritance of gene expression states and their regulators at the single-cell level.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

Quantitating CD8+ T cell memory development.

In acute immune responses to infection, memory T cells develop that can spawn recall responses. This process has not been observable directly in vivo. Here we highlight the utility of mathematical inference to derive quantitatively testable models of mammalian CD8+ T cell memory development from complex experimental data. Previous inference studies suggested that precursors of memory T cells arise early during the immune response. Recent work has both validated a crucial prediction of this T cell diversification model and refined the model. While multiple developmental routes to distinct memory subsets might exist, a branch point occurs early in proliferating T cell blasts, from which separate differentiation pathways emerge for slowly dividing precursors of re-expandable memory cells and rapidly dividing effectors.

Network switches and their role in circadian clocks.

Circadian rhythms are generated by complex interactions among genes and proteins. Self-sustained ∼24 h oscillations require negative feedback loops and sufficiently strong nonlinearities that are the product of molecular and network switches. Here, we review common mechanisms to obtain switch-like behavior, including cooperativity, antagonistic enzymes, multisite phosphorylation, positive feedback, and sequestration. We discuss how network switches play a crucial role as essential components in cellular circadian clocks, serving as integral parts of transcription-translation feedback loops that form the basis of circadian rhythm generation. The design principles of network switches and circadian clocks are illustrated by representative mathematical models that include bistable systems and negative feedback loops combined with Hill functions. This work underscores the importance of negative feedback loops and network switches as essential design principles for biological oscillations, emphasizing how an understanding of theoretical concepts can provide insights into the mechanisms generating biological rhythms.

The population context is a driver of the heterogeneous response of epithelial cells to interferons.

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.

Probing the range of applicability of structure- and energy-adjusted QM/MM link bonds III: QM/MM MD simulations of solid-state systems at the example of layered carbon structures.

The previously introduced workflow to achieve an energetically and structurally optimized description of frontier bonds in quantum mechanical/molecular mechanics (QM/MM)-type applications was extended into the regime of computational material sciences at the example of a layered carbon model systems. Optimized QM/MM link bond parameters at HSEsol/6-311G(d,p) and self-consistent density functional tight binding (SCC-DFTB) were derived for graphitic systems, enabling detailed investigation of specific structure motifs occurring in graphene-derived structures v i a quantum-chemical calculations. Exemplary molecular dynamics (MD) simulations in the isochoric-isothermic (NVT) ensemble were carried out to study the intercalation of lithium and the properties of the Stone-Thrower-Wales defect. The diffusivity of lithium as well as hydrogen and proton adsorption on a defective graphene surface served as additional example. The results of the QM/MM MD simulations provide detailed insight into the applicability of the employed link-bond strategy when studying intercalation and adsorption properties of graphitic materials.

Individual T helper cells have a quantitative cytokine memory.

The probabilistic expression of cytokine genes in differentiated T helper (Th) cell populations remains ill defined. By single-cell analyses and mathematical modeling, we show that one stimulation featured stable cytokine nonproducers as well as stable producers with wide cell-to-cell variability in the magnitude of expression. Focusing on interferon-γ (IFN-γ) expression by Th1 cells, mathematical modeling predicted that this behavior reflected different cell-intrinsic capacities and not mere gene-expression noise. In vivo, Th1 cells sort purified by secreted IFN-γ amounts preserved a quantitative memory for both probability and magnitude of IFN-γ re-expression for at least 1 month. Mechanistically, this memory resulted from quantitatively distinct transcription of individual alleles and was controlled by stable expression differences of the Th1 cell lineage-specifying transcription factor T-bet. Functionally, Th1 cells with graded IFN-γ production competence differentially activated Salmonella-infected macrophages for bacterial killing. Thus, individual Th cells commit to produce distinct amounts of a given cytokine, thereby generating functional intrapopulation heterogeneity.

Host-Guest Interactions of Ruthenium(II) Arene Complexes with Cucurbit[7/8]uril.

Cucurbit[n]urils (CB[n]s) have been recognized for their chemical and thermal stability, and their ability to bind many neutral and cationic guest molecules makes them excellent hosts in a range of supramolecular applications. In drug delivery, CB[n]s can enhance drug solubility, improve chemical and physical drug stability, and allow for triggered and controlled release. This study aimed to investigate the ability of CB[7] and CB[8] as molecular hosts to bind ruthenium(II) arene complexes that are current anticancer lead structures in the area of metallodrugs. Both, experimental and computational methods, led to insights into the binding preferences and geometries of [RuII(cym)Cl2]2 (1; cym = η6-p-cymene), [RuII(cym)(dmb)Cl2]) (2; cym = η6-p-cymene; dmb = 1,3-dimethylbenzimidazol-2-ylidene), and [RuII(cym)(pta)Cl2] (3, RAPTA-C; cym = η6-p-cymene; pta = 1,3,5-triaza-7-phospha-adamantane) with CB[7] and CB[8]. Competition experiments by mass spectrometry revealed clear preferences of 2 for CB[8] and 3 for CB[7]. Based on a comparison of the associated interaction energies from quantum chemical calculations as well as experimental data, 3@CB[7] clearly prefers a binding mode, where the pta ligand is located inside the cavity of the host, and the metal ion interacts with two of the carbonyl groups on the rim of CB[7]. In contrast, complex 2 binds in two different orientations with interaction energies similar to those of both CB[n]s, with the cym ligand being either inside or outside of the cavity. These findings suggest that ruthenium(II) arene complexes are able to form stable host-guest interactions with CB[n]s, which can be exploited as drug delivery vehicles in further metallodrug development to improve their chemical stability.

Towards hybrid quantum mechanical/molecular mechanical simulations of Li and Na intercalation in graphite - force field development and DFTB parametrisation.

In this work a previously established QM/MM simulation protocol for the treatment of solid-state interfaces was extended towards the treatment of layered bulk materials enabling for instance investigation of metal intercalation in graphitic carbon materials. In order to study the intercalation of Li in graphite, new density functional tight binding (DFTB) parameters for Li have been created. Molecular dynamics (MD) simulations at constant temperatures (273.15, 298.15 and 323.15 K) have been carried out to assess the performance of the presented DFTB MD simulation approach. The intercalation of variable lithium and sodium content was investigated via z-distribution functions and analysis of the diffusivity in the direction parallel to the graphene plane. Both the calculated diffusion coefficients and the activation energy in case of lithium are in good agreement with experimental data. The comparison of the QM/MM MD simulation results provide detailed insights into the structural and dynamical properties of intercalated metal ions.

Modelling bulk and surface characteristics of cubic CeO2, Gd2O3, and gadolinium-doped ceria using a partial charge framework.

The development and characterization of materials for solid oxide fuel cells (SOFC) is an important step towards sustainable energy technologies. This present study models cubic CeO2, Gd2O3, and gadolinium-doped ceria (GDC) using newly constructed interaction potentials based on a partial atom charge framework. The interaction model was validated by comparing the structural properties with experimental reference data, which were found to be in good agreement. Validation of the potential model was conducted considering the surface stability of CeO2 and Gd2O3. Additionally, the accuracy of the novel potential model was assessed by comparing the oxygen diffusion coefficient in GDCn (n = 4-15) and the associated activation energy. The results demonstrate that the novel potential model is capable of describing the oxygen diffusion in GDC. In addition, this study compares the vibrational properties of the bulk with density functional theory (DFT) calculations, using a harmonic frequency analysis that avoids the need for computationally expensive quantum mechanical molecular dynamics (QM MD) simulations. The potential is compatible with a reactive water model, thus providing a framework for the simulation of solid-liquid interfaces.

The impact of alcohol and ammonium fluoride on pressure-induced amorphization of cubic structure I clathrate hydrates.

We have investigated pressure-induced amorphization (PIA) of an alcohol clathrate hydrate (CH) of cubic structure type I (sI) in the presence of NH4F utilizing dilatometry and x-ray powder diffraction. PIA occurs at 0.98 GPa at 77 K, which is at a much lower pressure than for other CHs of the same structure type. The amorphized CH also shows remarkable resistance against crystallization upon decompression. While amorphized sI CHs could not be recovered previously at all, this is possible in the present case. By contrast to other CHs, the recovery of the amorphized CHs to ambient pressure does not even require a high-pressure annealing step, where recovery without any loss of amorphicity is possible at 120 K and below. Furthermore, PIA is accessible upon compression at unusually high temperatures of up to 140 K, where it reaches the highest degree of amorphicity. Molecular dynamics simulations confirm that polar alcoholic guests, as opposed to non-polar guests, induce cage deformation at lower pressure. The substitution of NH4F into the host-lattice stabilizes the collapsed state more than the crystalline state, thereby enhancing the collapse kinetics and lowering the pressure of collapse.