Cell-intrinsic Changes Research Articles

Cancer neuroscience at the brain-body interface.

Our approaches toward understanding cancer have evolved beyond cell-intrinsic and local microenvironmental changes within the tumor to encompass how the cancer interfaces with the entire host organism. The nervous system is uniquely situated at the interface between the brain and body, constantly receiving and sending signals back and forth to maintain homeostasis and respond to salient stimuli. It is becoming clear that various cancers disrupt this dialog between the brain and body via both neuronal and humoral routes, leading to aberrant brain activity and accelerated disease. In this outlook, I discuss this view of cancer as a homeostatic challenge, emphasize cutting-edge work, and provide outstanding questions that need to be answered to move the field forward.

Development of an epigenetic clock resistant to changes in immune cell composition

Epigenetic clocks are age predictors that use machine-learning models trained on DNA CpG methylation values to predict chronological or biological age. Increases in predicted epigenetic age relative to chronological age (epigenetic age acceleration) are connected to aging-associated pathologies, and changes in epigenetic age are linked to canonical aging hallmarks. However, epigenetic clocks rely on training data from bulk tissues whose cellular composition changes with age. Here, we found that human naive CD8+ T cells, which decrease in frequency during aging, exhibit an epigenetic age 15–20 years younger than effector memory CD8+ T cells from the same individual. Importantly, homogenous naive T cells isolated from individuals of different ages show a progressive increase in epigenetic age, indicating that current epigenetic clocks measure two independent variables, aging and immune cell composition. To isolate the age-associated cell intrinsic changes, we created an epigenetic clock, the IntrinClock, that did not change among 10 immune cell types tested. IntrinClock shows a robust predicted epigenetic age increase in a model of replicative senescence in vitro and age reversal during OSKM-mediated reprogramming.

Alzheimer’s disease rewires gene coexpression networks coupling different brain regions

Connectome studies have shown how Alzheimer’s disease (AD) disrupts functional and structural connectivity among brain regions. But the molecular basis of such disruptions is less studied, with most genomic/transcriptomic studies performing within-brain-region analyses. To inspect how AD rewires the correlation structure among genes in different brain regions, we performed an Inter-brain-region Differential Correlation (Inter-DC) analysis of RNA-seq data from Mount Sinai Brain Bank on four brain regions (frontal pole, superior temporal gyrus, parahippocampal gyrus and inferior frontal gyrus, comprising 264 AD and 372 control human post-mortem samples). An Inter-DC network was assembled from all pairs of genes across two brain regions that gained (or lost) correlation strength in the AD group relative to controls at FDR 1%. The differentially correlated (DC) genes in this network complemented known differentially expressed genes in AD, and likely reflects cell-intrinsic changes since we adjusted for cell compositional effects. Each brain region used a distinctive set of DC genes when coupling with other regions, with parahippocampal gyrus showing the most rewiring, consistent with its known vulnerability to AD. The Inter-DC network revealed master dysregulation hubs in AD (at genes ZKSCAN1, SLC5A3, RCC1, IL17RB, PLK4, etc.), inter-region gene modules enriched for known AD pathways (synaptic signaling, endocytosis, etc.), and candidate signaling molecules that could mediate region-region communication. The Inter-DC network generated in this study is a valuable resource of gene pairs, pathways and signaling molecules whose inter-brain-region functional coupling is disrupted in AD, thereby offering a new perspective of AD etiology.

Whole-body irradiation impairs naïve CD4 T cell function

Abstract Advancements in nuclear power and radiotherapy have expanded new avenues for whole-body irradiation (WBI) to humans, urging the need to understand its impact on immune cells. While most studies focus on the numerical recovery of naïve T cells post-WBI, less is known regarding its impact on their function. We subjected B6 SPF mice to 0/5Gy doses of WBI and used I-Ab tetramer enrichment to track the recovery of naïve CD4s of different antigen specificities post WBI. The number of MOG40 specific CD4 T cells is diminished 10 days post-irradiation (DPR), and recovered by 40DPR. To test if they are able to induce EAE post-WBI, we administered MOG+CFA, followed by pertussis toxin (PTX). The 10DPR mice showed delayed EAE development, likely due to diminished MOG40 naïve precursors. Interestingly, the 40DPR mice potentiated EAE compared to the 0Gy mice, evidenced by increased CD4 priming in the dLN, and spine infiltration. While WBI-mediated CD4 T cell-intrinsic changes could contribute to the timing/severity of EAE, T cell-extrinsic factors could also influence disease. To test if WBI compromises the blood-brain barrier (BBB) and facilitates EAE progression, EAE is induced in +/-WBI mice without PTX. In the absence of PTX, 0Gy mice did not develop EAE, while the 10- and 40DPR mice developed EAE suggesting BBB impairment. Overall, WBI directly impacts the number and function of autoreactive CD4 T cells, while helping them enter the spine by changing the environment.

Exposure to Mycobacterium remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Transcriptional Profiling of Skeletal Muscle Stem Cells After In Vivo Engraftment into a Heterologous Niche Environment.

Adult stem cells play a critical role in the maintenance and repair of the organs in which they reside. However, their function is highly dependent on the crosstalk with their niche environment that changes during development and in disease states. The niche provides signals to stem cells to activate, proliferate, self-renew, or remain in quiescence. In skeletal muscle, the niche is perturbed in disease contexts such as aging, muscular dystrophies, and cachexia. Therefore, it is important to develop methods that permit the decoupling of niche-mediated from cell-intrinsic changes that occur in muscle stem cells (MuSCs) in development and disease contexts. With the purpose of determining the effect of the niche environment on the MuSC transcriptome, function, or health, we have coupled an allogeneic stem cell transplantation system, meaning the transplantation of MuSCs from a donor mouse into a recipient host mouse, with Switching Mechanism at 5' End of RNA Template (SMART-Seq) to quantify the effects of the niche on the MuSC transcriptome in vivo. Briefly, MuSCs are isolated from a GFP reporter donor mouse (Pax7-nGFP) and transplanted into the irradiated muscles of immunocompromised allogeneic hosts. The MuSCs are re-isolated by fluorescence-activated cell sorting (FACS) after three weeks of inhabiting the heterologous niche, defined as a niche that is different from their originating niche, and sequencing-ready libraries are created. This method allows for the direct comparison of the transcriptome of stem cells before and after transplantation into a host of a different age, disease status, or genetic background. This method can be used to accurately quantify the direct effect of the niche environment on the stem cell gene expression profile and to decouple cell-intrinsic versus niche-mediated alterations in the stem cell transcriptome. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Allogeneic muscle stem cell transplantation.

The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression

AbstractMyelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

Targeting lymphoid-derived IL-17 signaling to delay skin aging

Skin aging is characterized by structural and functional changes that contribute to age-associated frailty. This probably depends on synergy between alterations in the local niche and stem cell-intrinsic changes, underscored by proinflammatory microenvironments that drive pleotropic changes. The nature of these age-associated inflammatory cues, or how they affect tissue aging, is unknown. Based on single-cell RNA sequencing of the dermal compartment of mouse skin, we show a skew towards an IL-17-expressing phenotype of T helper cells, γδ T cells and innate lymphoid cells in aged skin. Importantly, in vivo blockade of IL-17 signaling during aging reduces the proinflammatory state of the skin, delaying the appearance of age-related traits. Mechanistically, aberrant IL-17 signals through NF-κB in epidermal cells to impair homeostatic functions while promoting an inflammatory state. Our results indicate that aged skin shows signs of chronic inflammation and that increased IL-17 signaling could be targeted to prevent age-associated skin ailments.

Abstract 4117: Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies

Abstract T Cell Bispecific Antibodies (TCBs) are potent molecules that induce durable complete remissions with a favorable safety profile in Relapsed Refractory Non Hodgkin Lymphoma patients. Despite the promising clinical efficacy, a proportion of patients escape under treatment due to the occurrence of primary and acquired resistance mechanisms that may involve the modulation of target antigen expression, cancer cell intrinsic mechanisms, T cell functionality or treatment-induced changes in the tumor microenvironment (TME). Therefore, a better understanding of TCB-induced resistance mechanisms is of great importance to enable the design of novel therapeutic approaches and/or combination strategies to overcome resistance. We present the results of exploratory preclinical analysis using an aggressive lymphoma model in humanized mice and a syngeneic melanoma model in immunocompetent mice, focusing on the assessment of T cell functionality, TME reprogramming, and cancer cell intrinsic changes upon longitudinal TCB treatments, encompassing tumor regression and escape. Flow cytometry, IHC, scRNAseq and ex vivo functional analysis of T cell cytotoxicity and cytokine release demonstrate that intra-tumor T cell functionality is preserved over serial TCB treatments, including tumor escape. On the other hand, TCB treatment induced a reprogramming of the TME consisting of rapid myeloid cell infiltration, followed by the expansion of intra-tumor T cells at a later time point (favoring higher CD8:CD4 ratio). Cumulative evidence from in vitro and in vivo scRNAseq experiments provided insights into macrophage reaction to TCB-mediated T cell inflammation, consisting of a rapid (as early as 2 h) activation and upregulation of several pro-inflammatory molecules (IL-1b, CXCL8, CXCL9, CXCL10, CXCL1, CCL2, CCL3, CCL4, CCL5, IFN-g-induced genes: GBP2 -8 and BATF2). This was followed by a shift towards a more suppressive phenotype at later time points (20-96 h) as evidenced by the expression of PD-L1, INHBA, IDO, FLT1 genes. The analysis of cancer cells showed upregulation of Wnt/beta catenin, p53 and Hedgehog signaling pathways at tumor escape. In contrast, proliferation signatures including MYC, E2F target genes, MTORC signaling, and G2M checkpoint, along with metabolic pathways of oxidative phosphorylation and glycolysis were enriched in cancer cells at regression. Taken together, our data show that T cell cytotoxicity and functional states are preserved during tumor regression and tumor escape phases, suggesting that T cell dysfunctionality is unlikely to be the sole cause of TCB resistance. TME reprogramming, consisting of a rapid myeloid cell infiltration and their acquisition of a suppressive phenotype over time, along with a selection of cancer cell clones that escape the TCB-mediated cytotoxicity and upregulate Wnt, p53 and Hedgehog pathways could contribute to TCB-mediated resistance, leading to tumor escape. Citation Format: Johannes Sam, Llucia Alberti Servera, Sina Nassiri, Atul Sethi, Alison Ribeiro, Gabrielle Leclercq-Cohen, Petra Schwalie, Tamara Hüsser, Emilio Yangueez, Sylvia Herter, Christian Klein, Pablo Umana, Marina Bacac. Molecular insights into resistance mechanisms to therapy with T cell bispecific antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4117.

IL-6-targeted therapies to block the cytokine or its receptor drive distinct alterations in T cell function.

Therapeutics that inhibit IL-6 at different points in its signaling pathway are in clinical use, yet whether the immunological effects of these interventions differ based on their molecular target is unknown. We performed short-term interventions in individuals with type 1 diabetes using anti-IL-6 (siltuximab) or anti-IL-6 receptor (IL-6R; tocilizumab) therapies and investigated the impact of this in vivo blockade on T cell fate and function. Immune outcomes were influenced by the target of the therapeutic intervention (IL-6 versus IL-6R) and by peak drug concentration. Tocilizumab reduced ICOS expression on T follicular helper cell populations and T cell receptor-driven (TCR-driven) STAT3 phosphorylation. Siltuximab reversed resistance to Treg-mediated suppression and increased TCR-driven phosphorylated STAT3 and production of IL-10, IL-21, and IL-27 by T effectors. Together, these findings indicate that the context of IL-6 blockade in vivo drives distinct T cell-intrinsic changes that may influence therapeutic outcomes.

Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive. Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential. To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin-/cKitlo/Sca1lo/Flk2+/IL-7Rα+) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1S291fs-transduced WT and Mlkl-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS. Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

Abstract 1096: Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy

Abstract Aggressive lung cancers such as non-small cell lung carcinomas (NSCLC) commonly recur following first line therapy. Recurring tumors metastasize more rapidly. We hypothesize that drug-resistant phenotypes created by the selection pressure of first line treatments such as platinum-based cytotoxic agents contribute to the increased aggressivity of recurring NSCLC. We showed previously that an engineered model of cisplatin-resistant NSCLC tissues secretes more inflammatory cytokines compared to untreated controls and induces more severe muscle tissue injury in a microphysiological model of cachexia processes. Here, we report methods for the systematic generation of cisplatin-resistant A549 lung adenocarcinoma cell cultures and demonstrate their increased aggressivity in 3D spheroid culture and in 3D organotypic culture with patient-derived lung cancer-associated fibroblasts (CAF). We treated A549 cells with cisplatin for 96 hours at doses of 15-45µM, yielding cultures of 54.8-8.9% surviving cells as quantified via flow cytometric of cell size and permeability. Here we report characterization of spheroids and tissues composed of cells selected for resistance with 25 µM cisplatin (Cis-R) compared with untreated parent A549. This dose was selected because we were unable to recover proliferating cultures with lower percentages of surviving. Cis-R spheroids exhibited a significant reduction in circularity that is indicative of increased asymmetry. We measured a Ki67 index of 6.4 +/- 1.4% for parent A549 spheroids and 49.7 +/- 9.5% for Cis-R spheroids. The marked increase in cell proliferation of CisR spheroids correlated with increased distances of invasion and more mesenchymal-like cell morphologies of invading cells in 3D cultures of Cis-R spheroids without CAF. These data suggest that cisplatin resistant A549 created using our protocol are more proliferative and invasive in 3D culture environments due to cell intrinsic changes. Organotypic 3D cultures of Cis-R spheroids and CAF exhibited an increased density and alignment of myofibroblast-like CAF with smooth muscle actin cytoskeleton when compared to cultures of parent spheroids with the same CAF. Our results demonstrate proof of principle for modeling recurrent tumors with more aggressive phenotypes using this approach. Future experiments will translate these methods to patient-derived cultures and test the hypothesis that cisplatin-resistant recurrent NSCLC stimulates more severe local and distant stromal reactions as a driver of metastasis. Engineering validated models of drug-resistant recurring cancers will help accelerate the discovery of anti-metastasis therapies in human cell-based microphysiological systems. Citation Format: Omar M. Ahmad, Elisabet A. Olsen, Delia A. Carlino, Charles E. Byrne, Mark Mondrinos. Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1096.

Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells

Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.

Intrinsic excitability mechanisms of neuronal ensemble formation.

Neuronal ensembles are coactive groups of cortical neurons, found in spontaneous and evoked activity, that can mediate perception and behavior. To understand the mechanisms that lead to the formation of ensembles, we co-activated layer 2/3 pyramidal neurons in brain slices from mouse visual cortex, in animals of both sexes, replicating in vitro an optogenetic protocol to generate ensembles in vivo. Using whole-cell and perforated patch-clamp pair recordings we found that, after optogenetic or electrical stimulation, coactivated neurons increased their correlated activity, a hallmark of ensemble formation. Coactivated neurons showed small biphasic changes in presynaptic plasticity, with an initial depression followed by a potentiation after a recovery period. Optogenetic and electrical stimulation also induced significant increases in frequency and amplitude of spontaneous EPSPs, even after single-cell stimulation. In addition, we observed unexpected strong and persistent increases in neuronal excitability after stimulation, with increases in membrane resistance and reductions in spike threshold. A pharmacological agent that blocks changes in membrane resistance reverted this effect. These significant increases in excitability can explain the observed biphasic synaptic plasticity. We conclude that cell-intrinsic changes in excitability are involved in the formation of neuronal ensembles. We propose an 'iceberg' model, by which increased neuronal excitability makes subthreshold connections suprathreshold, enhancing the effect of already existing synapses, and generating a new neuronal ensemble.

Malnutrition Disrupts T Cell Migration

Abstract Malnutrition is associated with reductions in the number and function of T lymphocytes. Previous studies in the lab suggest that malnutrition may also impart a “super-quiescent” phenotype to T cells, perhaps affecting the efficiency of their migration within and between lymph nodes. Thus, the purpose of this study is to evaluate the effect of malnutrition on T cell migration in vivo and to characterize malnutrition-induced changes in the expression of proteins known to be important for T cell migration. To determine if malnourishment alters T cell migration in vivo, we compared lymph node entry rates of adoptively-transferred malnourished and control T cells in malnourished and control recipients. In agreement with other studies, control CD4+ T cells were more efficient than control CD8+ T cells at entering the lymph nodes. Interestingly, regardless of recipient diet, malnourished CD4+ and CD8+ T cells entered the lymph nodes at equivalent rates, suggesting that malnourishment eliminates distinct lymph node entry efficiencies for CD8+ and CD4+ T cells. We also found important differences in the expression of key proteins involved in T cell migration between malnourished and control mice. Overall, we found that malnutrition disrupts T cell migration including the distinct migration efficiencies of CD4+ and CD8+ T cells. An improved understanding of T cell-intrinsic changes that occur during malnourishment should enhance our knowledge of CD4+ and CD8+ T cell migration and shed light on how organisms adapt to malnutrition. Supported by NSF-MRI [DBI- 1920116] NSF-RUI [IOS-1951881]

Intermediate Filaments in Cellular Mechanoresponsiveness: Mediating Cytoskeletal Crosstalk From Membrane to Nucleus and Back.

The mammalian cytoskeleton forms a mechanical continuum that spans across the cell, connecting the cell surface to the nucleus via transmembrane protein complexes in the plasma and nuclear membranes. It transmits extracellular forces to the cell interior, providing mechanical cues that influence cellular decisions, but also actively generates intracellular forces, enabling the cell to probe and remodel its tissue microenvironment. Cells adapt their gene expression profile and morphology to external cues provided by the matrix and adjacent cells as well as to cell-intrinsic changes in cytoplasmic and nuclear volume. The cytoskeleton is a complex filamentous network of three interpenetrating structural proteins: actin, microtubules, and intermediate filaments. Traditionally the actin cytoskeleton is considered the main contributor to mechanosensitivity. This view is now shifting owing to the mounting evidence that the three cytoskeletal filaments have interdependent functions due to cytoskeletal crosstalk, with intermediate filaments taking a central role. In this Mini Review we discuss how cytoskeletal crosstalk confers mechanosensitivity to cells and tissues, with a particular focus on the role of intermediate filaments. We propose a view of the cytoskeleton as a composite structure, in which cytoskeletal crosstalk regulates the local stability and organization of all three filament families at the sub-cellular scale, cytoskeletal mechanics at the cellular scale, and cell adaptation to external cues at the tissue scale.

Decline of regenerative potential of old muscle stem cells: contribution to muscle aging.

Muscle stem cells (MuSCs) are required for life-long muscle regeneration. In general, aging has been linked to a decline in the numbers and the regenerative potential of MuSCs. Muscle regeneration depends on the proper functioning of MuSCs, which is itself dependent on intricate interactions with its niche components. Aging is associated with both cell-intrinsic and niche-mediated changes, which can be the result of transcriptional, posttranscriptional, or posttranslational alterations in MuSCs or in the components of their niche. The interplay between cell intrinsic alterations in MuSCs and changes in the stem cell niche environment during aging and its impact on the number and the function of MuSCs is an important emerging area of research. In this review, we discuss whether the decline in the regenerative potential of MuSCs with age is the cause or the consequence of aging skeletal muscle. Understanding the effect of aging on MuSCs and the individual components of their niche is critical to develop effective therapeutic approaches to diminish or reverse the age-related defects in muscle regeneration.

Sepsis leads to lasting changes in phenotype and function of memory CD8 T cells.

The global health burden due to sepsis and the associated cytokine storm is substantial. While early intervention has improved survival during the cytokine storm, those that survive can enter a state of chronic immunoparalysis defined by transient lymphopenia and functional deficits of surviving cells. Memory CD8 T cells provide rapid cytolysis and cytokine production following re-encounter with their cognate antigen to promote long-term immunity, and CD8 T cell impairment due to sepsis can pre-dispose individuals to re-infection. While the acute influence of sepsis on memory CD8 T cells has been characterized, if and to what extent pre-existing memory CD8 T cells recover remains unknown. Here, we observed that central memory CD8 T cells (TCM) from septic patients proliferate more than those from healthy individuals. Utilizing LCMV immune mice and a CLP model to induce sepsis, we demonstrated that TCM proliferation is associated with numerical recovery of pathogen-specific memory CD8 T cells following sepsis-induced lymphopenia. This increased proliferation leads to changes in composition of memory CD8 T cell compartment and altered tissue localization. Further, memory CD8 T cells from sepsis survivors have an altered transcriptional profile and chromatin accessibility indicating long-lasting T cell intrinsic changes. The sepsis-induced changes in the composition of the memory CD8 T cell pool and transcriptional landscape culminated in altered T cell function and reduced capacity to control L. monocytogenes infection. Thus, sepsis leads to long-term alterations in memory CD8 T cell phenotype, protective function and localization potentially changing host capacity to respond to re-infection.

Preservation of satellite cell number and regenerative potential with age reveals locomotory muscle bias

BackgroundAlthough muscle regenerative capacity declines with age, the extent to which this is due to satellite cell-intrinsic changes vs. environmental changes has been controversial. The majority of aging studies have investigated hindlimb locomotory muscles, principally the tibialis anterior, in caged sedentary mice, where those muscles are abnormally under-exercised.MethodsWe analyze satellite cell numbers in 8 muscle groups representing locomotory and non-locomotory muscles in young and 2-year-old mice and perform transplantation assays of low numbers of hind limb satellite cells from young and old mice.ResultsWe find that satellite cell density does not decline significantly by 2 years of age in most muscles, and one muscle, the masseter, shows a modest but statistically significant increase in satellite cell density with age. The tibialis anterior and extensor digitorum longus were clear exceptions, showing significant declines. We quantify self-renewal using a transplantation assay. Dose dilution revealed significant non-linearity in self-renewal above a very low threshold, suggestive of competition between satellite cells for space within the pool. Assaying within the linear range, i.e., transplanting fewer than 1000 cells, revealed no evidence of decline in cell-autonomous self-renewal or regenerative potential of 2-year-old murine satellite cells.ConclusionThese data demonstrate the value of comparative muscle analysis as opposed to overreliance on locomotory muscles, which are not used physiologically in aging sedentary mice, and suggest that self-renewal impairment with age is precipitously acquired at the geriatric stage, rather than being gradual over time, as previously thought.

T cell dysfunction in Activated PI3K δ Syndrome explained by cell intrinsic and extrinsic components

Abstract Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many T cell receptors and has been implicated in the control of CD4+ T cell helper differentiation, activation and proliferation. APDS patients have increased PI3K activity that leads to several immune manifestations including lymphoproliferation, Th2-related pathologies and impaired Ab responses. Analysis of a CRISPR/Cas9 mouse model (Pik3cdE1020K) showed a decreased frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation, as well as changes in cytokine expression and T cell help. Our studies investigated CD4+ T cell intrinsic and extrinsic alterations contributing to the dysregulation of the immune response. We identified cell intrinsic features resulting in increased IL-4 and IL-5, explaining the Th2 mediated disease in these patients. Additionally, cell intrinsic changes in Tfh generation and function were shown in the in vivo mouse model, contributing to an impaired ability to sustain GCs and alter isotype switching. Furthermore, additional studies have shown that the overactive T cells result from both intrinsic and extrinsic factors, with contributions from both dysregulated T cells and B cells exerting effects on T cell activation. Together, these studies reveals an important role of PI3K in modulating T cell differentiation and function through both T cell-intrinsic and extrinsic mechanisms. This provides new insights into the APDS pathophysiology with potential to improve the clinical management of these patients.