Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

Regulatory T cells suppress Th17 cell Ca2+ signaling in the spinal cord during murine autoimmune neuroinflammation.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

Regulatory T cells promote remyelination in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis following human neural stem cell transplant.

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that affects the central nervous system (CNS) for which there is no cure. In MS, encephalitogenic T cells infiltrate the CNS causing demyelination and neuroinflammation; however, little is known about the role of regulatory T cells (Tregs) in CNS tissue repair. Transplantation of neural stem and progenitor cells (NSCs and NPCs) is a promising therapeutic strategy to promote repair through cell replacement, although recent findings suggest transplanted NSCs also instruct endogenous repair mechanisms. We have recently described that dampened neuroinflammation and increased remyelination is correlated with emergence of Tregs following human NPC transplantation in a murine viral model of immune-mediated demyelination. In the current study we utilized the prototypic murine autoimmune model of demyelination experimental autoimmune encephalomyelitis (EAE) to test the efficacy of hNSC transplantation. Eight-week-old, male EAE mice receiving an intraspinal transplant of hNSCs during the chronic phase of disease displayed remyelination, dampened neuroinflammation, and an increase in CNS CD4+CD25+FoxP3+ regulatory T cells (Tregs). Importantly, ablation of Tregs abrogated histopathological improvement. Tregs are essential for maintenance of T cell homeostasis and prevention of autoimmunity, and an emerging role for Tregs in maintenance of tissue homeostasis through interactions with stem and progenitor cells has recently been suggested. The data presented here provide direct evidence for collaboration between CNS Tregs and hNSCs promoting remyelination.

Sialylation may be dispensable for reciprocal modulation of helper T cells by intravenous immunoglobulin.

Several mechanisms account for the beneficial effect of intravenous immunoglobulin (IVIg) in autoimmune and inflammatory diseases. These mechanisms include effects on the cellular compartment and on the humoral compartment. Thus, IVIg impacts on dendritic cells, macrophages, neutrophils, basophils, NK cells, and B and T lymphocytes. Several studies have emphasized that the antiinflammatory effect of IVIg is dependent on α2,6-sialylation of the N-linked glycan on asparagine-297 of the Fc portion of IgG. However, recent reports have questioned the necessity of sialylated Fc and the role of FcγRIIB in IVIg-mediated antiinflammatory effects. In view of the critical role played by Th17 cells in several autoimmune pathologies and the increasing use of IVIg in several of these conditions, by using neuraminidase-treated, desialylated IVIg, we addressed whether the α2,6-sialylation of IgG is essential for the beneficial effect of IVIg in experimental autoimmune encephalomyelitis (EAE), a Th17-driven condition, and for the reciprocal modulation of helper T-cell subsets. We observed no difference in the ability of IVIg to ameliorate EAE irrespective of its sialylation. Our findings thus show that sialylation of IVIg is not necessary for IVIg-mediated amelioration of EAE or for downregulation of Th17 cells and upregulation of regulatory T cells.

Intravenous gammaglobulin inhibits encephalitogenic potential of pathogenic T cells and interferes with their trafficking to the central nervous system, implicating sphingosine-1 phosphate receptor 1-mammalian target of rapamycin axis.

Despite an increasing use of high-dose therapy of i.v. gammaglobulin (IVIg) in the treatment of various T cell- and Ab-mediated inflammatory and autoimmune diseases, comprehension of the mechanisms underlying its therapeutic benefit has remained a major challenge. Particularly, the effect of IVIg in T cell-mediated autoimmune conditions remains unexplored. Using an actively induced experimental autoimmune encephalomyelitis model, a T cell-mediated autoimmune condition, we demonstrate that IVIg inhibits the differentiation of naive CD4 T cells into encephalitogenic subsets (Th1 and Th17 cells) and concomitantly induces an expansion of Foxp3(+) regulatory T cells. Further, IVIg renders effector T cells less pathogenic by decreasing the expression of encephalitogenic molecular players like GM-CSF and podoplanin. Intriguingly and contrary to the current arguments, the inhibitory FcγRIIB is dispensable for IVIg-mediated reciprocal modulation of effector and regulatory CD4 subsets. Additionally, F(ab')2 fragments also retained this function of IVIg. IVIg or F(ab')2 fragments decrease the sphingosine-1 phosphate receptor on CD4 cells, thus sequestering these cells in the draining lymph nodes and decreasing their infiltration into the CNS. Our study reveals a novel role of Igs in the modulation of polarization and trafficking of T lymphocytes, accounting for the observed beneficial effect in IVIg therapy.

Protocol to quantify the activation dynamics of tumor-associated T cells in mice by functional intravital microscopy.

Tumor-associated T cells orchestrate cancer rejection after checkpoint blockade immunotherapy. T cell function depends on dynamic antigen recognition through the T cell receptor (TCR) resulting in T cell activation. Here, we present an approach to quantify the dynamics and magnitude of tumor-associated T cell activation at multiple time points in living mice using the genetically encoded calcium reporter Salsa6f and functional intravital microscopy (F-IVM). Our protocol allows researchers to measure the activation dynamics of various immune cells in vivo. For complete details on the use and execution of this protocol, please refer to Geels et al.1.

Immune Checkpoint Inhibition-related Neuroinflammation Disrupts Cognitive Function.

Combinatorial blockade of Cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and Programmed Cell Death Protein 1 (PD-1) significantly improve the progression-free survival of individuals with metastatic cancers, including melanoma. In addition to unleashing anti-tumor immunity, combination immune checkpoint inhibition (ICI) disrupts immune-regulatory networks critical for maintaining homeostasis in various tissues, including the central nervous system (CNS). Although ICI- and cancer-related cognitive impairments (CRCI) in survivors are increasingly becoming evident, our understanding of ICI-induced immune-related adverse effects (IREA) in the CNS remains incomplete. Here, our murine melanoma model reveals that combination ICI impairs hippocampal-dependent learning and memory, as well as memory consolidation processes. Mechanistically, combination ICI disrupted synaptic integrity, and neuronal plasticity, reduced myelin, and further predisposed CNS for exaggerated experimental autoimmune encephalomyelitis. Combination ICI substantially altered both lymphoid and myeloid cells in the CNS. Neurogenesis was unaffected, however, microglial activation persisted for two-months post- ICI, concurrently with cognitive deficits, which parallels clinical observations in survivors. Overall, our results demonstrate that blockade of CTLA-4 and PD-1 alters neuro-immune homeostasis and activates microglia, promoting long-term neurodegeneration and driving cognitive impairments. Therefore, limiting microglial activation is a potential avenue to mitigate CNS IRAE while maintaining the therapeutic benefits of rapidly evolving ICIs and their combinations. SIGNIFICANCE: Despite the superior therapeutic efficacy of immune checkpoint inhibition (ICI) for cancers, its undesired effects on brain function are not fully understood. Here, we demonstrate that combination ICI elevates neuroinflammation, activates microglia, leading to detrimental neurodegenerative and neurocognitive sequelae.

Polymodal K+ channel modulation contributes to dual analgesic and anti-inflammatory actions of traditional botanical medicines.

Pain and inflammation contribute immeasurably to reduced quality of life, yet modern analgesic and anti-inflammatory therapeutics can cause dependence and side effects. Here, we screened 1444 plant extracts, prepared primarily from native species in California and the United States Virgin Islands, against two voltage-gated K+ channels - T-cell expressed Kv1.3 and nociceptive-neuron expressed Kv7.2/7.3. A subset of extracts both inhibits Kv1.3 and activates Kv7.2/7.3 at hyperpolarized potentials, effects predicted to be anti-inflammatory and analgesic, respectively. Among the top dual hits are witch hazel and fireweed; polymodal modulation of multiple K+ channel types by hydrolysable tannins contributes to their dual anti-inflammatory, analgesic actions. In silico docking and mutagenesis data suggest pore-proximal extracellular linker sequence divergence underlies opposite effects of hydrolysable tannins on different Kv1 isoforms. The findings provide molecular insights into the enduring, widespread medicinal use of witch hazel and fireweed and demonstrate a screening strategy for discovering dual anti-inflammatory, analgesic small molecules.

Interruption of the intratumor CD8+ T cell:Treg crosstalk improves the efficacy of PD-1 immunotherapy.

PD-1 blockade unleashes potent antitumor activity in CD8+ T cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen the response to immunotherapy. Tumor-Treg inhibition is a promising strategy to improve the efficacy of checkpoint blockade immunotherapy; however, our understanding of the mechanisms supporting tumor-Tregs during PD-1 immunotherapy is incomplete. Here, we show that PD-1 blockade increases tumor-Tregs in mouse models of melanoma and metastatic melanoma patients. Mechanistically, Treg accumulation is not caused by Treg-intrinsic inhibition of PD-1 signaling but depends on an indirect effect of activated CD8+ T cells. CD8+ T cells produce IL-2 and colocalize with Tregs in mouse and human melanomas. IL-2 upregulates the anti-apoptotic protein ICOS on tumor-Tregs, promoting their accumulation. Inhibition of ICOS signaling before PD-1 immunotherapy improves control over immunogenic melanoma. Thus, interrupting the intratumor CD8+ T cell:Treg crosstalk represents a strategy to enhance the therapeutic efficacy of PD-1 immunotherapy.

Interruption of the Intratumor CD8:Treg Crosstalk Improves the Efficacy of PD-1 Immunotherapy.

PD-1 blockade unleashes the potent antitumor activity of CD8 cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen response to immunotherapy. Tumor Treg inhibition is a promising strategy to overcome therapeutic resistance; however, the mechanisms supporting tumor Tregs during PD-1 immunotherapy are largely unexplored. Here, we report that PD-1 blockade increases tumor Tregs in mouse models of immunogenic tumors, including melanoma, and metastatic melanoma patients. Unexpectedly, Treg accumulation was not caused by Treg-intrinsic inhibition of PD-1 signaling but instead depended on an indirect effect of activated CD8 cells. CD8 cells colocalized with Tregs within tumors and produced IL-2, especially after PD-1 immunotherapy. IL-2 upregulated the anti-apoptotic protein ICOS on tumor Tregs, causing their accumulation. ICOS signaling inhibition before PD-1 immunotherapy resulted in increased control of immunogenic melanoma. Thus, interrupting the intratumor CD8:Treg crosstalk is a novel strategy that may enhance the efficacy of immunotherapy in patients.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Effect of IVIg on human dendritic cell-mediated antigen uptake and presentation: role of lipid accumulation.

Intravenous immunoglobulin (IVIg) is a therapeutic preparation consisting of pools of normal, polyspecific IgG antibodies obtained from plasma of several thousand healthy individuals. In addition to its use in primary and secondary immune deficiency, IVIg is increasingly used in the therapy of a large number of autoimmune conditions. Despite its successful use in immunopathologies for over two decades, the precise mechanisms underlying the therapeutic benefit have not been fully elucidated. We and others have demonstrated that IVIg inhibits the antigen uptake and presentation by dendritic cells (DC). Here we report that IVIg-mediated inhibition of uptake and processing of antigens is associated with an increased accumulation of lipid as analyzed by flow cytometry and electron microscopy. As accumulation of lipids in DC is known to impart tolerogenic properties, these findings unravel novel link between antibodies and intracellular physiology of innate cells and may further uncover novel immunoregulatory mechanisms of IVIg in auto-inflammatory diseases.

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses.

Several mental illnesses, characterized by aberrant stress reactivity, often arise after early-life adversity (ELA). However, it is unclear how ELA affects stress-related brain circuit maturation, provoking these enduring vulnerabilities. We find that ELA increases functional excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, resulting from disrupted developmental synapse pruning by adjacent microglia. Microglial process dynamics and synaptic element engulfment were attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor MerTK. Accordingly, selective chronic chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Notably, selective early-life activation of ELA microglia normalized adult acute and chronic stress responses, including stress-induced hormone secretion and behavioral threat responses, as well as chronic adrenal hypertrophy of ELA mice. Thus, microglial actions during development are powerful contributors to mechanisms by which ELA sculpts the connectivity of stress-regulating neurons, promoting vulnerability to stress and stress-related mental illnesses.

Mechanically activated ion channel Piezo1 modulates macrophage polarization and stiffness sensing.

Macrophages perform diverse functions within tissues during immune responses to pathogens and injury, but molecular mechanisms by which physical properties of the tissue regulate macrophage behavior are less well understood. Here, we examine the role of the mechanically activated cation channel Piezo1 in macrophage polarization and sensing of microenvironmental stiffness. We show that macrophages lacking Piezo1 exhibit reduced inflammation and enhanced wound healing responses. Additionally, macrophages expressing the transgenic Ca2+ reporter, Salsa6f, reveal that Ca2+ influx is dependent on Piezo1, modulated by soluble signals, and enhanced on stiff substrates. Furthermore, stiffness-dependent changes in macrophage function, both in vitro and in response to subcutaneous implantation of biomaterials in vivo, require Piezo1. Finally, we show that positive feedback between Piezo1 and actin drives macrophage activation. Together, our studies reveal that Piezo1 is a mechanosensor of stiffness in macrophages, and that its activity modulates polarization responses.

Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.

Immunomodulation by intravenous immunoglobulin: role of regulatory T cells.

An altered immune homeostasis as a result of deficiency or defective function of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) is common in several autoimmune diseases. Hence, therapeutic strategies to render Tregs functionally competent are being investigated. Intravenous immunoglobulin (IVIG) is being increasingly used for the treatment of a wide range of autoimmune and inflammatory diseases. Recent studies have demonstrated that IVIG induces the expansion of Tregs and enhances their suppressive functions. These effects of IVIG on Tregs correlate with the beneficial effects of IVIG in patients with autoimmune diseases. Thus, modulation of Tregs by IVIG represents a novel mode of action that explains the therapeutic effects of IVIG in T cell-mediated autoimmune diseases. However, the molecular mechanisms involved in IVIG-mediated modulation of Tregs are unclear and need further investigation.

Calcium Dynamics in Astrocytes During Cell Injury.

The changes in intracellular calcium concentration ([Ca2+]) following laser-induced cell injury in nearby cells were studied in primary mouse astrocytes selectively expressing the Ca2+ sensitive GFAP-Cre Salsa6f fluorescent tandem protein, in an Ast1 astrocyte cell line, and in primary mouse astrocytes loaded with Fluo4. Astrocytes in these three systems exhibit distinct changes in [Ca2+] following induced death of nearby cells. Changes in [Ca2+] appear to result from release of Ca2+ from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca2+ changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca2+] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.