Identification of aberrant luminal progenitors and mTORC1 as a potential breast cancer prevention target in BRCA2 mutation carriers.

Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2mut/+ tissue, including expansion of aberrant ERBB3lo luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3lo progenitors in BRCA2mut/+ breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3lo progenitors could generate both ER+ and ER- cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.

Programmed death receptor 1 (PD-1) ligand Fc fusion proteins reduce T-cell proliferation in vitro independently of PD-1.

Programmed death receptor 1 (PD-1) is an inhibitory receptor on T cells shown to restrain T-cell proliferation. PD-1 immune checkpoint blockade has emerged as a highly promising approach in cancer treatment. Much of our understanding of the function of PD-1 is derived from in vitro T-cell activation assays. Here we set out to further investigate how T cells integrate inhibitory signals such as PD-1 in vitro using the PD-1 agonist, PD-1 ligand 1 (PD-L1) fusion protein (PD-L1.Fc), coimmobilized alongside anti-CD3 agonist monoclonal antibody (mAb) on plates to deliver PD-1 signals to wild-type and PD-1-/- CD8+ T cells. Surprisingly, we found that the PD-L1.Fc fusion protein inhibited T-cell proliferation independently of PD-1. This PD-L1.Fc inhibition was observed in the presence and absence of CD28 and interleukin-2 signaling. Binding of PD-L1.Fc was restricted to PD-1-expressing T cells and thus inhibition was not mediated by the interaction of PD-L1.Fc with CD80 or other yet unknown binding partners. Furthermore, a similar PD-1-independent reduction of T-cell proliferation was observed with plate-bound PD-L2.Fc. Hence, our results suggest that the coimmobilization of PD-1 ligand fusion proteins with anti-CD3 mAb leads to a reduction of T-cell engagement with plate-bound anti-CD3 mAb. This study demonstrates a nonspecific mechanism of T-cell inhibition when PD-L1.Fc or PD-L2.Fc fusion proteins are delivered in a plate-bound coimmobilization assay and highlights the importance of careful optimization of assay systems and reagents when interpreting their influence on T-cell proliferation.

Extracellular matrix proteoglycans support aged hippocampus networks: a potential cellular-level mechanism of brain reserve.

One hallmark of normative brain aging is vast heterogeneity in whether older people succumb to or resist cognitive decline. Resilience describes a brain's capacity to maintain cognition in the face of aging and disease. One factor influencing resilience is brain reserve-the status of neurobiological resources available to support neuronal circuits as dysfunction accumulates. This study uses a cohort of behaviorally characterized adult, middle-aged, and aged rats to test whether neurobiological factors that protect inhibitory neurotransmission and synapse function represent key components of brain reserve. Histochemical analysis of extracellular matrix proteoglycans, which play critical roles in stabilizing synapses and modulating inhibitory neuron excitability, was conducted alongside analyses of lipofuscin-associated autofluorescence. The findings indicate that aging results in lower proteoglycan density and more lipofuscin in CA3. Aged rats with higher proteoglycan density exhibited better performance on the Morris watermaze, whereas lipofuscin abundance was not related to spatial memory. These data suggest that the local environment around neurons may protect against synapse dysfunction or hyperexcitability and could contribute to brain reserve mechanisms.

Expansion of MAIT cells in the combined absence of NKT and γδ-T cells.

Mucosal-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and γδT cells are collectively referred to as 'unconventional T cells' due to their recognition of non-peptide antigens and restriction to MHC-I-like molecules. However, the factors controlling their widely variable frequencies between individuals and organs are poorly understood. We demonstrated that MAIT cells are increased in NKT or γδT cell-deficient mice and highly expand in mice lacking both cell types. TCRα repertoire analysis of γδT cell-deficient thymocytes revealed altered Trav segment usage relative to wild-type thymocytes, highlighting retention of the Tcra-Tcrd locus from the 129 mouse strain used to generate Tcrd-/- mice. This resulted in a moderate increase in distal Trav segment usage, including Trav1, potentially contributing to increased generation of Trav1-Traj33+ MAIT cells in the Tcrd-/- thymus. Importantly, adoptively transferred MAIT cells underwent increased homeostatic proliferation within NKT/gdT cell-deficient tissues, with MAIT cell subsets exhibiting tissue-specific homing patterns. Our data reveal a shared niche for unconventional T cells, where competition for common factors may be exploited to collectively modulate these cells in the immune response. Lastly, our findings emphasise careful assessment of studies using NKT or γδT cell-deficient mice when investigating the role of unconventional T cells in disease.

Early immune pressure initiated by tissue-resident memory T cells sculpts tumor evolution in non-small cell lung cancer.

Tissue-resident memory T (TRM) cells provide immune defense against local infection and can inhibit cancer progression. However, it is unclear to what extent chronic inflammation impacts TRM activation and whether TRM cells existing in tissues before tumor onset influence cancer evolution in humans. We performed deep profiling of healthy lungs and lung cancers in never-smokers (NSs) and ever-smokers (ESs), finding evidence of enhanced immunosurveillance by cells with a TRM-like phenotype in ES lungs. In preclinical models, tumor-specific or bystander TRM-like cells present prior to tumor onset boosted immune cell recruitment, causing tumor immune evasion through loss of MHC class I protein expression and resistance to immune checkpoint inhibitors. In humans, only tumors arising in ES patients underwent clonal immune evasion, unrelated to tobacco-associated mutagenic signatures or oncogenic drivers. These data demonstrate that enhanced TRM-like activity prior to tumor development shapes the evolution of tumor immunogenicity and can impact immunotherapy outcomes.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential.

The αß and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αß and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

Overexpression of Lmo2 initiates T-lymphoblastic leukemia via impaired thymocyte competition.

Cell competition has recently emerged as an important tumor suppressor mechanism in the thymus that inhibits autonomous thymic maintenance. Here, we show that the oncogenic transcription factor Lmo2 causes autonomous thymic maintenance in transgenic mice by inhibiting early T cell differentiation. This autonomous thymic maintenance results in the development of self-renewing preleukemic stem cells (pre-LSCs) and subsequent leukemogenesis, both of which are profoundly inhibited by restoration of thymic competition or expression of the antiapoptotic factor BCL2. Genomic analyses revealed the presence of Notch1 mutations in pre-LSCs before subsequent loss of tumor suppressors promotes the transition to overt leukemogenesis. These studies demonstrate a critical role for impaired cell competition in the development of pre-LSCs in a transgenic mouse model of T cell acute lymphoblastic leukemia (T-ALL), implying that this process plays a role in the ontogeny of human T-ALL.

SCI Thrive: Impact of a peer-led online self-management program.

OBJECTIVE: To test the effectiveness of a peer-led online self-management program for individuals with spinal cord injury (SCI). DESIGN: Randomized waitlist control trial. SETTING: Community. PARTICIPANTS: 184 adults with SCI. INTERVENTIONS: SCI Thrive is a peer-led self-management program with self-paced online content and video-sessions for live discussion. OUTCOME MEASURES: Perceived quality of life, self-efficacy for health, participation. RESULTS: A total of 97 individuals (86 randomized plus 9 assigned to the final group) were assigned to treatment and 86 randomized to the waitlist. Participants were 51 years old on average (SD = 14.9), with 58% male, mean of 15.5 (SD = 14.0) years injured, with 59% cervical injuries and 64% incomplete injuries. The treatment group had significantly higher scores on CHART occupational subscale (P = .022), but no other differences were found at the end of 6 weeks. Analysis of all participants who completed SCI Thrive showed significant increase in self-efficacy between baseline (6.32) and 6 weeks (6.81; P < .001) which was maintained at 3 months post treatment (6.83; P = .001). Those who were more engaged in SCI Thrive reported higher quality of life (P = .001), self-efficacy (P = .007), and increased mobility on the CHART (P = .026). CONCLUSION: SCI Thrive is a highly accessible program for individuals with SCI and shows promise for improving self-efficacy. Strategies to increase engagement should be added to maximize benefits. Measurement tools may have been impacted by COVID-19 pandemic. Further research on the efficacy of SCI Thrive is needed given feedback on benefit of a group focus area, such as on physical activity.

Dietary tryptophan deficiency promotes gut RORγt+ Treg cells at the expense of Gata3+ Treg cells and alters commensal microbiota metabolism.

Micronutrient deficiency is a major cause of disease throughout the world. Yet, how perturbations influence the immune-microbiome interface remains poorly understood. Here, we report that loss of dietary tryptophan (Trp) reshapes intestinal microbial communities, including the depletion of probiotic L. reuteri, drives transcriptional changes to immune response genes in the intestinal ileum, and reshapes the regulatory T cell (Treg) compartment. Dietary Trp deficiency promotes expansion of RORγt+ Treg cells and the loss of Gata3+ Tregs in a microbiota-dependent manner. In the absence of dietary Trp, provision of the AhR ligand indole-3-carbinol is sufficient to restore the Treg compartment. Together, these data show that dietary Trp deficiency perturbs the interaction between the host and its bacterial symbionts to regulate Treg homeostasis via the deprivation of bacterially derived Trp metabolites. Our findings highlight an essential role for immune-microbiome crosstalk as a key homeostatic regulator during nutrient deficiency.

Retrosplenial cortex microglia and perineuronal net densities are associated with memory impairment in aged rhesus macaques.

Synapse loss and altered plasticity are significant contributors to memory loss in aged individuals. Microglia, the innate immune cells of the brain, play critical roles in maintaining synapse function, including through a recently identified role in regulating the brain extracellular matrix. This study sought to determine the relationship between age, microglia, and extracellular matrix structure densities in the macaque retrosplenial cortex. Twenty-nine macaques ranging in age from young adult to aged were behaviorally characterized on 3 distinct memory tasks. Microglia, parvalbumin (PV)-expressing interneurons and extracellular matrix structures, known as perineuronal nets (PNNs), were immuno- and histochemically labeled. Our results indicate that microglia densities increase in the retrosplenial cortex of aged monkeys, while the proportion of PV neurons surrounded by PNNs decreases. Aged monkeys with more microglia had fewer PNN-associated PV neurons and displayed slower learning and poorer performance on an object recognition task. Stepwise regression models using age and the total density of aggrecan, a chondroitin sulfate proteoglycan of PNNs, better predicted memory performance than did age alone. Together, these findings indicate that elevated microglial activity in aged brains negatively impacts cognition in part through mechanisms that alter PNN assembly in memory-associated brain regions.

Venetoclax treatment in patients with cancer has limited impact on circulating T and NK cells.

Venetoclax is an effective treatment for certain blood cancers, such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). However, most patients relapse while on venetoclax and further treatment options are limited. Combining venetoclax with immunotherapies is an attractive approach; however, a detailed understanding of how venetoclax treatment impacts normal immune cells in patients is lacking. In this study, we performed deep profiling of peripheral blood (PB) cells from patients with CLL and AML before and after short-term treatment with venetoclax using mass cytometry (cytometry by time of flight) and found no impact on the concentrations of key T-cell subsets or their expression of checkpoint molecules. We also analyzed PB from patients with breast cancer receiving venetoclax long-term using a single-cell multiomics approach (cellular indexing of transcriptomes and epitopes by sequencing) and functional assays. We found significant depletion of B-cell populations with low expression of MCL-1 relative to other immune cells, attended by extensive transcriptomic changes. By contrast, there was less impact on circulating T cells and natural killer (NK) cells, with no changes in their subset composition, transcriptome, or function following venetoclax treatment. Our data indicate that venetoclax has minimal impact on circulating T or NK cells, supporting the rationale of combining this BH3 mimetic drug with cancer immunotherapies for more durable antitumor responses.

Single-Cell Profiling of the Intrinsic Apoptotic Pathway by Mass Cytometry (CyTOF).

Mass cytometry time-of-flight (CyTOF) is a technology for the study of complex biological processes at the single-cell level. The technology enables measurement of >50 protein moieties on the surface and inside the cell. The power of CyTOF lies in the application of purpose-built panels of antibody probes that resolve features of key biological processes in a cell. Here, we describe this technology's use to profile changes in the intrinsic apoptotic (cell death) protein machinery at a single-cell level. We provide a comprehensive overview of a tailor-made set of cell survival/death antibodies, ideal staining conditions, and high-dimensional data analysis.

Location, location, location! Rewiring IL-2 circuits defines context-specific outcomes.

Here, we highlight a recent publication by Whyte et al. that reveals diverse immune outcomes of interleukin (IL)-2 expression in distinct microenvironments. Their definition of context-dependent IL-2 networks paves the way for the development of tissue-specific therapies that enlist the potent immuno-modulatory activity of IL-2.

Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy.

Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.

Meta-prospective memory accuracy in young adults with and without depressive symptoms.

Prospective memory (PM) is the ability to remember an intention in the future. Individuals with depression are candidates for PM failures, resulting in detrimental consequences, such as forgetting to take a medication or implement therapeutic techniques; inaccurate assessments of one's PM abilities can exacerbate these issues. The current study examined if appraisals about one's prospective memory (meta-PM) performance differs between healthy and depressed adults. Data were gathered from 137 adults and included self-reported depression, PM beliefs, objective PM, and assessment of executive functions (EFs). Participants were separated into depressed/healthy categories based on a self-report measure. There was a non-significant correlation between self-reported PM and objective PM for both depressed (r = .06, p = .61) and healthy (r = .08, p = .52) groups, suggesting both groups had inaccurate meta-PM. There were non-significant differences in meta-PM between these groups (Fisher's Z = -0.09, p = .93), but exploratory gender analyses revealed women's meta-PM was significantly less accurate than men's. Women had higher reports of depression and PM complaints compared to men. This study lends evidence that depression is not necessarily related to worse meta-PM accuracy, despite depression's association with memory complaints, and that women are at greater risk for inaccurate meta-PM.

Aged Rats Exhibit Altered Behavior-Induced Oscillatory Activity, Place Cell Firing Rates, and Spatial Information Content in the CA1 Region of the Hippocampus.

Hippocampal gamma and theta oscillations are associated with mnemonic and navigational processes and adapt to changes in the behavioral state of an animal to optimize spatial information processing. It has been shown that locomotor activity modulates gamma and theta frequencies in rats, although how age alters this modulation has not been well studied. Here, we examine gamma and theta local-field potential and place cell activity in the hippocampus CA1 region of young and old male rats as they performed a spatial eye-blink conditioning task across 31 d. Although mean gamma frequency was similar in both groups, gamma frequency increased with running speed at a slower rate in old animals. By contrast, theta frequencies scaled with speed similarly in both groups but were lower across speeds in old animals. Although these frequencies scaled equally well with deceleration and speed, acceleration was less correlated with gamma frequency in both age groups. Additionally, spike phase-locking to gamma, but not theta, was greater in older animals. Finally, aged rats had reduced within-field firing rates but greater spatial information per spike within the field. These data support a strong relationship between locomotor behavior and local-field potential activity and suggest that age significantly affects this relationship. Furthermore, observed changes in CA1 place cell firing rates and information content lend support to the hypothesis that age may result in more general and context-invariant hippocampal representations over more detailed information. These results may explain the observation that older adults tend to recall the gist of an experience rather than the details.SIGNIFICANCE STATEMENT Hippocampal oscillations and place cell activity are sensitive to sensorimotor input generated from active locomotion, yet studies of aged hippocampal function often do not account for this. By considering locomotion and spatial location, we identify novel age-associated differences in the scaling of oscillatory activity with speed, spike-field coherence, spatial information content, and within-field firing rates of CA1 place cells. These results indicate that age has an impact on the relationship between locomotion and hippocampal oscillatory activity, perhaps indicative of alterations to afferent input. These data also support the hypothesis that aged hippocampal place cells, compared with young, may more often represent more general spatial information. If true, these results may help explain why older humans tend to recall less specific and more gist-like information.

Microglia Drive Pockets of Neuroinflammation in Middle Age.

During aging, microglia produce inflammatory factors, show reduced tissue surveillance, altered interactions with synapses, and prolonged responses to CNS insults, positioning these cells to have profound impact on the function of nearby neurons. We and others recently showed that microglial attributes differ significantly across brain regions in young adult mice. However, the degree to which microglial properties vary during aging is largely unexplored. Here, we analyze and manipulate microglial aging within the basal ganglia, brain circuits that exhibit prominent regional microglial heterogeneity and where neurons are vulnerable to functional decline and neurodegenerative disease. In male and female mice, we demonstrate that VTA and SNc microglia exhibit unique and premature responses to aging, compared with cortex and NAc microglia. This is associated with localized VTA/SNc neuroinflammation that may compromise synaptic function as early as middle age. Surprisingly, systemic inflammation, local neuron death, and astrocyte aging do not appear to underlie these early aging responses of VTA and SNc microglia. Instead, we found that microglial lysosome status was tightly linked to early aging of VTA microglia. Microglial ablation/repopulation normalized VTA microglial lysosome swelling and suppressed increases in VTA microglial density during aging. In contrast, CX3CR1 receptor KO exacerbated VTA microglial lysosome rearrangements and VTA microglial proliferation during aging. Our findings reveal a previously unappreciated regional variation in onset and magnitude of microglial proliferation and inflammatory factor production during aging and highlight critical links between microglial lysosome status and local microglial responses to aging.SIGNIFICANCE STATEMENT Microglia are CNS cells that are equipped to regulate neuronal health and function throughout the lifespan. We reveal that microglia in select brain regions begin to proliferate and produce inflammatory factors in late middle age, months before microglia in other brain regions. These findings demonstrate that CNS neuroinflammation during aging is not uniform. Moreover, they raise the possibility that local microglial responses to aging play a critical role in determining which populations of neurons are most vulnerable to functional decline and neurodegenerative disease.

PD-1 cooperates with AIRE-mediated tolerance to prevent lethal autoimmune disease.

Immunological tolerance is established and maintained by a diverse array of safeguards that work together to protect against autoimmunity. Despite the identification of numerous tolerogenic processes, the basis for cooperation among them remains poorly understood. We sought to identify synergy among several well-defined tolerance mediators that alone provide protection only from mild autoimmune symptoms in C57BL/6 mice: BIM, AIRE, CBL-B, and PD-1. Survey of a range of compound mutant mice revealed that the combined loss of the autoimmune regulator, AIRE, with PD-1 unleashed a spontaneous, lethal autoimmune disease. Pdcd1−/−Aire−/− mice succumbed to cachexia before adulthood, with near-complete destruction of the exocrine pancreas. Such fatal autoimmunity was not observed in Pdcd1−/−Bim−/−, Bim−/−Aire−/−, or Cblb−/−Bim−/− mice, suggesting that the cooperation between AIRE-mediated and PD-1­mediated tolerance was particularly potent. Immune profiling revealed largely normal development of FOXP3+ regulatory T (Treg) cells in Pdcd1−/−Aire−/− mice, yet excessive, early activation of effector T cells. Adoptive transfer experiments demonstrated that autoimmune exocrine pancreatitis was driven by conventional CD4+ T cells and could not be prevented by the cotransfer of Treg cells from wild-type mice. The development of autoimmunity in mixed bone marrow chimeras supported these observations, indicating that failure of recessive tolerance was responsible for disease. These findings reveal a potent tolerogenic axis between AIRE and PD-1 that has implications for our understanding of how immune checkpoint blockade might synergize with subclinical defects in central tolerance to elicit autoimmune disease.

Caspase-8 has dual roles in regulatory T cell homeostasis balancing immunity to infection and collateral inflammatory damage.

Targeting the potent immunosuppressive properties of FOXP3+ regulatory T cells (Tregs) has substantial therapeutic potential for treating autoimmune and inflammatory diseases. Yet, the molecular mechanisms controlling Treg homeostasis, particularly during inflammation, remain unclear. We report that caspase-8 is a central regulator of Treg homeostasis in a context-specific manner that is decisive during immune responses. In mouse genetic models, targeting caspase-8 in Tregs led to accumulation of effector Tregs resistant to apoptotic cell death. Conversely, inflammation induced the MLKL-dependent necroptosis of caspase-8-deficient lymphoid and tissue Tregs, which enhanced immunity to a variety of chronic infections to promote clearance of viral or parasitic pathogens. However, improved immunity came at the risk of lethal inflammation in overwhelming infections. Caspase-8 inhibition using a clinical-stage compound revealed that human Tregs have heightened sensitivity to necroptosis compared with conventional T cells. These findings reveal a fundamental mechanism in Tregs that could be targeted to manipulate the balance between immune tolerance versus response for therapeutic benefit.