Hallmarks of tumor-experienced T cells are absent in multiple myeloma patients from diagnosis through maintenance therapy.

Dysregulation of the bone marrow (BM) niche in multiple myeloma (MM) alters the composition and state of resident immune cells, potentially impeding anti-tumor immunity. One common mechanism of immune inhibition in solid tumors is the induction of exhaustion in tumor-specific T cells. However, the extent of T cell tumor recognition and exhaustion is not well-characterized in MM. As the specific mechanisms of immune evasion are critical for devising effective therapeutic strategies, we deeply profiled the CD8+ T cell compartment of newly-diagnosed MM (NDMM) patients for evidence of tumor reactivity and T cell exhaustion. We applied single-cell multi-omic sequencing and antigen-specific mass cytometry to longitudinal BM and peripheral blood (PB) samples taken from timepoints spanning from diagnosis through induction therapy, autologous stem cell transplant (ASCT), and maintenance therapy. We identified an exhausted-like population that lacked several canonical exhaustion markers, was not significantly enriched in NDMM patients, and consisted of small, nonpersistent clones. We also observed an activated population with increased frequency in the PB of NDMM patients exhibiting phenotypic and clonal features consistent with homeostatic, antigen-nonspecific activation. However, there was no evidence of "tumor-experienced" T cells displaying hallmarks of terminal exhaustion and/or tumor-specific activation/expansion in NDMM patients at any timepoint.

Bi-directional neuro-immune dysfunction after chronic experimental brain injury.

BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in murine bone development.

Fetal bone development occurs through the conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescues angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.

The brain-bone marrow axis and its implications for chronic traumatic brain injury.

Traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function which contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham congenic donor mice into otherwise healthy, age-matched, irradiated hosts. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI showed that longer reconstitution periods were associated with increased microgliosis and leukocyte infiltration. Thus, TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in innate immunity and neurological function, as well as altered sensitivity to subsequent brain injury.

Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

Persistent serum protein signatures define an inflammatory subcategory of long COVID.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.

Lipid-Polymer Hybrid Nanoparticles Utilize B Cells and Dendritic Cells to Elicit Distinct Antigen-Specific CD4+ and CD8+ T Cell Responses.

Antigen-presenting cells (APCs) are widely studied for treating immune-mediated diseases, and dendritic cells (DCs) are potent APCs that uptake and present antigens (Ags). However, DCs face several challenges that hinder their clinical translation due to their inability to control Ag dosing and low abundance in peripheral blood. B cells are a potential alternative to DCs, but their poor nonspecific Ag uptake capabilities compromise controllable priming of T cells. Here, we developed phospholipid-conjugated Ags (L-Ags) and lipid-polymer hybrid nanoparticles (L/P-Ag NPs) as delivery platforms to expand the range of accessible APCs for use in T cell priming. These delivery platforms were evaluated using DCs, CD40-activated B cells, and resting B cells to understand the impacts of various Ag delivery mechanisms for generation of Ag-specific T cell responses. L-Ag delivery (termed depoting) of MHC class I- and II-restricted Ags successfully loaded all APC types in a tunable manner and primed both Ag-specific CD8+ and CD4+ T cells, respectively. Incorporating L-Ags and polymer-conjugated Ags (P-Ag) into NPs can direct Ags to different uptake pathways to engineer the dynamics of presentation and shape T cell responses. DCs were capable of processing and presenting Ag delivered from both L- and P-Ag NPs, yet B cells could only utilize Ag delivered from L-Ag NPs, which led to differential cytokine secretion profiles in coculture studies. Altogether, we show that L-Ags and P-Ags can be rationally paired within a single NP to leverage distinct delivery mechanisms to access multiple Ag processing pathways in two APC types, offering a modular delivery platform for engineering Ag-specific immunotherapies.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration.

Lipofuscin is an autofluorescent (AF) pigment formed by lipids and misfolded proteins, which accumulates in postmitotic cells with advanced age. Here, we immunophenotyped microglia in the brain of old C57BL/6 mice (>18 months old) and demonstrate that in comparison to young mice, one-third of old microglia are AF, characterized by profound changes in lipid and iron content, phagocytic activity, and oxidative stress. Pharmacological depletion of microglia in old mice eliminated the AF microglia following repopulation and reversed microglial dysfunction. Age-related neurological deficits and neurodegeneration after traumatic brain injury (TBI) were attenuated in old mice lacking AF microglia. Furthermore, increased phagocytic activity, lysosomal burden, and lipid accumulation in microglia persisted for up to 1 year after TBI, were modified by APOE4 genotype, and chronically driven by phagocyte-mediated oxidative stress. Thus, AF may reflect a pathological state in aging microglia associated with increased phagocytosis of neurons and myelin and inflammatory neurodegeneration that can be further accelerated by TBI.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF-ERK1/2 signaling pathways.

A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF-ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.

Targeted Delivery of Chloroquine to Antigen-Presenting Cells Enhances Inhibition of the Type I Interferon Response.

Systemic lupus erythematosus (SLE) causes damaging inflammation in multiple organs via the accumulation of immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) via toll-like receptors (TLRs), contributing to disease pathogenesis by driving the secretion of inflammatory type I interferons (IFNs). Antimalarial drugs, such as chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE. However, they require ∼3 months of continuous use before achieving therapeutic efficacy and can accumulate in the retinal pigment epithelium with chronic use, resulting in retinopathy. We hypothesized that poly(ethylene glycol)-b-poly(propylene sulfide) filamentous nanocarriers, filomicelles (FMs), could directly deliver CQ to pDCs via passive, morphology-based targeting to concentrate drug delivery to specific immune cells, improve drug activity by increased inhibition of type I IFN, and enhance efficacy per dose. Healthy human peripheral blood mononuclear cells were treated with soluble CQ or CQ-loaded FMs, stimulated with TLR agonists or SLE patient sera, and type I IFN secretion was quantified via multi-subtype IFN-α ELISA and MX1 gene expression using real-time reverse transcription-quantitative polymerase chain reaction. Our results showed that 50 µg CQ/mg FM decreased MX1 expression and IFN-α production after TLR activation with either synthetic nucleic acid agonists or immune complex-rich sera from SLE patients. Cellular uptake and biodistribution studies showed that FMs preferentially accumulate in human pDCs and monocytes in vitro and in tissues frequently damaged in SLE patients (i.e., kidneys), while sparing the eye in vivo. These results showed that nanocarrier morphology enables drug delivery, and CQ-FMs may be equally effective and more targeted than soluble CQ at inhibiting SLE-relevant pathways.

Pathogenesis, Symptomatology, and Transmission of SARS-CoV-2 through Analysis of Viral Genomics and Structure.

The novel coronavirus SARS-CoV-2, which emerged in late 2019, has since spread around the world and infected hundreds of millions of people with coronavirus disease 2019 (COVID-19). While this viral species was unknown prior to January 2020, its similarity to other coronaviruses that infect humans has allowed for rapid insight into the mechanisms that it uses to infect human hosts, as well as the ways in which the human immune system can respond. Here, we contextualize SARS-CoV-2 among other coronaviruses and identify what is known and what can be inferred about its behavior once inside a human host. Because the genomic content of coronaviruses, which specifies the virus's structure, is highly conserved, early genomic analysis provided a significant head start in predicting viral pathogenesis and in understanding potential differences among variants. The pathogenesis of the virus offers insights into symptomatology, transmission, and individual susceptibility. Additionally, prior research into interactions between the human immune system and coronaviruses has identified how these viruses can evade the immune system's protective mechanisms. We also explore systems-level research into the regulatory and proteomic effects of SARS-CoV-2 infection and the immune response. Understanding the structure and behavior of the virus serves to contextualize the many facets of the COVID-19 pandemic and can influence efforts to control the virus and treat the disease. IMPORTANCE COVID-19 involves a number of organ systems and can present with a wide range of symptoms. From how the virus infects cells to how it spreads between people, the available research suggests that these patterns are very similar to those seen in the closely related viruses SARS-CoV-1 and possibly Middle East respiratory syndrome-related CoV (MERS-CoV). Understanding the pathogenesis of the SARS-CoV-2 virus also contextualizes how the different biological systems affected by COVID-19 connect. Exploring the structure, phylogeny, and pathogenesis of the virus therefore helps to guide interpretation of the broader impacts of the virus on the human body and on human populations. For this reason, an in-depth exploration of viral mechanisms is critical to a robust understanding of SARS-CoV-2 and, potentially, future emergent human CoVs (HCoVs).

Longitudinal immune dynamics of mild COVID-19 define signatures of recovery and persistence.

SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

Pathogenesis, Symptomatology, and Transmission of SARS-CoV-2 through Analysis of Viral Genomics and Structure.

The novel coronavirus SARS-CoV-2, which emerged in late 2019, has since spread around the world and infected hundreds of millions of people with coronavirus disease 2019 (COVID-19). While this viral species was unknown prior to January 2020, its similarity to other coronaviruses that infect humans has allowed for rapid insight into the mechanisms that it uses to infect human hosts, as well as the ways in which the human immune system can respond. Here, we contextualize SARS-CoV-2 among other coronaviruses and identify what is known and what can be inferred about its behavior once inside a human host. Because the genomic content of coronaviruses, which specifies the virus's structure, is highly conserved, early genomic analysis provided a significant head start in predicting viral pathogenesis and in understanding potential differences among variants. The pathogenesis of the virus offers insights into symptomatology, transmission, and individual susceptibility. Additionally, prior research into interactions between the human immune system and coronaviruses has identified how these viruses can evade the immune system's protective mechanisms. We also explore systems-level research into the regulatory and proteomic effects of SARS-CoV-2 infection and the immune response. Understanding the structure and behavior of the virus serves to contextualize the many facets of the COVID-19 pandemic and can influence efforts to control the virus and treat the disease.

Leveraging Heterogeneity in Systemic Lupus Erythematosus for New Therapies.

Systemic lupus erythematosus (SLE) is a multisystem, chronic autoimmune disease where treatment varies by patient and disease activity. Strong preclinical results and clinical correlates have motivated development of many drugs, but many of these have failed to achieve efficacy in clinical trials. FDA approval of belimumab in 2011 was the first successful SLE drug in nearly six decades. In this article, we review insights into the molecular and clinical heterogeneity of SLE from transcriptomics studies and detail their potential impact on drug development and clinical practices. We critically examine the pipeline of SLE drugs, including past failures and their associated lessons and current promising approaches. Finally, we identify opportunities for integrating these findings and drug development with new multidisciplinary advances to enhance future SLE treatment.

Impact of Four Common Hydrogels on Amyloid-ß (Aß) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer's Disease.

The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer's disease (AD) that could benefit from the in vivo-like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-ß (Aß), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aß cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aß is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aß structure and aggregation kinetics in solution and hydrogels composed of type I collagen, agarose, hyaluronic acid, and polyethylene glycol using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with enhanced Aß aggregation and Aß cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aß structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.

A Cell-Free Protein Expression System Derived from Human Primary Peripheral Blood Mononuclear Cells.

Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.

Lipid-Mediated Insertion of Toll-Like Receptor (TLR) Ligands for Facile Immune Cell Engineering.

Cell-based immunotherapies have tremendous potential to treat many diseases, such as activating immunity in cancer or suppressing it in autoimmune diseases. Most cell-based cancer immunotherapies in the clinic provide adjuvant signals through genetic engineering to enhance T cell functions. However, genetically encoded signals have minimal control over dosing and persist for the life of a cell lineage. These properties make it difficult to balance increasing therapeutic efficacy with reducing toxicities. Here, we demonstrated the potential of phospholipid-coupled ligands as a non-genetic system for immune cell engineering. This system provides simple, controlled, non-genetic adjuvant delivery to immune cells via lipid-mediated insertion into plasma membranes. Lipid-mediated insertion (termed depoting) successfully delivered Toll-like receptor (TLR) ligands intracellularly and onto cell surfaces of diverse immune cells. These ligands depoted into immune cells in a dose-controlled fashion and did not compete during multiplex pairwise loading. Immune cell activation could be enhanced by autocrine and paracrine mechanisms depending on the biology of the TLR ligand tested. Depoted ligands functionally persisted on plasma membranes for up to 4 days in naïve and activated T cells, enhancing their activation, proliferation, and skewing cytokine secretion. Our data showed that depoted ligands provided a persistent yet non-permanent adjuvant signal to immune cells that may minimize the intensity and duration of toxicities compared to permanent genetic delivery. Altogether, these findings demonstrate potential for lipid-mediated depoting as a universal cell engineering approach with unique, complementary advantages to other cell engineering methods.