Astrocytes lure CXCR2-expressing CD4+ T cells to gray matter via TAK1-mediated chemokine production in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system driven by peripheral immune cell infiltration and glial activation. The pathological hallmark of MS is demyelination, and mounting evidence suggests neuronal damage in gray matter is a major contributor to disease irreversibility. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Here, we used a modified EAE mouse model (Type-B EAE) that displays severe neuronal damage to investigate the interplay between peripheral immune cells and glial cells in the event of neuronal damage. We show that CD4⁺ T cells migrate to the spinal cord gray matter, preferentially to ventral horns. Compared to CD4⁺ T cells in white matter, gray matter-infiltrated CD4⁺ T cells were mostly immobilized and interacted with neurons, which are behaviors associated with detrimental effects to normal neuronal function. T cell-specific deletion of CXCR2 significantly decreased CD4⁺ T cell infiltration into gray matter in Type-B EAE mice. Further, astrocyte-targeted deletion of TAK1 inhibited production of CXCR2 ligands such as CXCL1 in gray matter, successfully prevented T cell migration into spinal cord gray matter, and averted neuronal damage and motor dysfunction in Type-B EAE mice. This study identifies astrocyte chemokine production as a requisite for the invasion of CD4⁺T cell into the gray matter to induce neuronal damage.

Multiple sclerosis (MS) is a prevalent, chronic neurologic autoimmune disease that results in accumulating disability. Disease onset usually occurs at 20–50 y of age and is characterized by symptoms of numbness, pain, fatigue, and/or visual impairment (1–3). Within 15–25 y of onset, 50% of MS patients require assistance with walking (4, 5) and 50% of MS patients report neurocognitive impairment (6). Accumulation of debilitating symptoms is attributed to an episodically inflamed central nervous system (CNS) as a result from recurrent attacks by immune cells (7).Demyelinated lesions are the classical hallmark of MS (8, 9); thus, the disease is historically considered a disease that primarily affects white matter of the CNS. In the past 20 y, mounting evidence suggested that inflammatory lesions in the CNS are not restricted to white matter but are also observed in CNS gray matter (9–11). In addition to myelin loss, gray matter lesions present with neuronal damage characterized by axonal transection, synaptic loss, and even neuronal loss (12–16). Neuronal damage is proposed to underlie the permanent and irreversible neurological dysfunctions in persons with MS (17, 18).The infiltration of antigen-specific lymphocytes such as T cells is implicated in CNS gray matter damage observed in MS (19) and an established mouse model, experimental autoimmune encephalomyelitis (EAE) (7–9). In the classical EAE model, T cells are mainly restricted to white matter of the spinal cord (20, 21) and are rarely found in spinal cord gray matter, with few exceptions (22). How T cells arise in CNS gray matter during MS pathogenesis is poorly understood. To mediate neuronal damage, T cells must be trafficked from lymphoid organs of peripheral tissues, such as lymph nodes and spleen, before transmigrating into the CNS. Such migration can occur via a vascular route through the blood–brain barrier, blood-cerebrospinal fluid, or meningeal lymphatic system (23, 24). Lymphocyte infiltration into the CNS is a tightly regulated process that is controlled by multiple factors that are cell-intrinsic or cell-extrinsic, including blood–brain barrier status, adhesion molecule expression, and presence of migratory cues (24, 25). During neuroinflammation, invading immune cells and local reactive glial cells create signaling gradients by secreting chemoattracting small peptide mediators to attract pathogenic cells to sites of inflammation. CNS-resident astrocytes have been identified as a key producer of important chemokines to induce the migration of T cells (26, 27).Here, while exploring the drivers of severe neuronal damage in spinal cord gray matter of mice induced to have a neurodegenerative form of EAE (termed Type-B EAE), we made the serendipitous observation that Type-B EAE is characterized by massive infiltration of CD4⁺ T cells into gray matter of the spinal cord. Accumulation of CD4⁺ T cells in spinal cord gray matter was prevented by genetic ablation of T cell CXCR2. Additionally, genetic ablation of astrocyte TAK1, an upstream molecule of CXCR2 ligand CXCL1, successfully prevented T cell migration to spinal cord gray matter, neuronal damage, and motor dysfunction.     

Pharmacological modulation of T cell immunity results in long-term remission of autoimmune arthritis

Chronic inflammatory diseases like rheumatoid arthritis are characterized by a deficit in fully functional regulatory T cells. DNA-methylation inhibitors have previously been shown to promote regulatory T cell responses and, in the present study, we evaluated their potential to ameliorate chronic and acute animal models of rheumatoid arthritis. Of the drugs tested, decitabine was the most effective, producing a sustained therapeutic effect that was dependent on indoleamine 2,3-dioxygenase (IDO) and was associated with expansion of induced regulatory T cells, particularly at the site of disease activity. Treatment with decitabine also caused apoptosis of Th1 and Th17 cells in active arthritis in a highly selective manner. The molecular basis for this selectivity was shown to be ENT1, a nucleoside transporter, which facilitates intracellular entry of the drug and is up-regulated on effector T cells during active arthritis. It was further shown that short-term treatment with decitabine resulted in the generation of a population of regulatory T cells that were able to suppress arthritis upon adoptive transfer. In summary, a therapeutic approach using an approved drug is described that treats active inflammatory disease effectively and generates robust regulatory T cells with the IDO-dependent capacity to maintain remission.

Despite recent advances in the therapy of chronic inflammatory diseases such as rheumatoid arthritis (RA), the induction of drug-free disease remission remains an elusive goal for the majority of patients. One reason for this is the lack of available drugs with the capacity to target pathogenic T effector (Teff) cell subsets selectively, while sparing or increasing the activity of regulatory T (Treg) cells.In health, Treg cells play a nonredundant role in maintaining self-tolerance by controlling the activity of Teff cells. However, chronic inflammation is associated with decreased Treg cell function, a phenomenon that has been documented in a number of autoimmune diseases, including RA, systemic lupus erythematosus, and type 1 diabetes (1–4). In the case of RA, we and others have previously shown that loss of Treg cell function is accompanied by reduced expression of CTLA-4 due to aberrant CpG methylation in the cis-regulatory regions of the gene (5). In addition, the importance of DNA demethylation at the FOXP3 locus in determining Treg cell function has been confirmed in a number of independent studies (6–12).These findings led us to question whether DNA-demethylating agents could promote Treg cell responses in chronic inflammatory diseases. There are two broad classes of DNA-demethylating agents: nucleoside analogs and nonnucleoside analogs. Two nucleoside analogs, decitabine and azacytidine, are approved for use in cancer due to their ability to induce cell death at high dose and promote reexpression of silenced tumor suppressor genes at low dose (13). Serendipitously, it was observed that numbers of circulating Treg cells are increased in the peripheral blood of patients following treatment with azacytidine (14–16). In addition, nucleoside analog demethylating agents have been reported to promote the generation and suppressive function of induced Treg (iTreg) cells in vitro (14, 17, 18) and to protect against experimental autoimmune encephalomyelitis and allogeneic cardiac transplant rejection in vivo (19).Against this background, we compared the ability of nucleoside- and nonnucleoside-based DNA-demethylating agents to promote induction of Treg cells in animal models of RA. We found that short-term treatment with the cytosine analog decitabine depleted pathogenic Teff cells and promoted Treg cell responses, leading to lasting disease remission.     

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate–BCoA–CPT1A axis for the regulation of immune homeostasis.

Regulatory T (Treg) cells are CD4⁺ T cells expressing Foxp3 that play a key role in immune suppression (1–3). They can be divided into natural Treg (nTreg) and inducible Treg (iTreg) cells (1–3). nTreg cells, which are often referred to as thymic Treg (tTreg), arise during CD4⁺ T cell differentiation in the thymus under the influence of relatively high-avidity interactions of the T cell receptor (TCR) with self-antigens (1–3). iTreg cells, also called peripherally induced Treg (pTreg), develop in secondary lymphoid tissues. In the presence of TGFβ1, naive CD4⁺ T are induced into iTreg cells upon TCR ligation and costimulation by antigen-presenting cells (APCs) in response to non-self antigens, such as allergens, food, and the commensal microbiota (1–3).It has been demonstrated that iTreg cells are enriched in gut-associated lymphoid tissues (GALTs) and are important for the maintenance of intestinal immune homeostasis (3–5). Intestinal iTreg cells were found to be important for the regulation of inflammatory bowel diseases (IBDs), such as Crohn’s disease (CD) and ulcerative colitis (UC), which can potentially affect any portion of the gastrointestinal tract and induce many further complications such as tissue fibrosis, stenosis, fistulas, and colon cancer over time (6). Enhancement of intestinal iTreg function or adoptive transfer of iTreg could significantly alleviate IBDs in mice (7–9).Different types of T cells are featured by distinct metabolic characteristics. Unlike effector CD4⁺ T cells (Teffs), including Th1, Th2, Th9, and Th17 cells, that are mainly reliant on aerobic glycolysis, iTreg cells largely rely on fatty acid oxidation (FAO) (10–12). Accumulating evidence has demonstrated that T cell differentiation is always coupled with metabolic reprogramming (13, 14). For instance, FAO needs to be established in the process of iTreg differentiation. Up-regulation of FAO improved iTreg generation, whereas impairment in FAO compromised iTreg differentiation (12, 13, 15, 16).FAO, comprised of a cyclical series of reactions, demands different fatty acids (FAs), which can be divided into long-, medium-, and short-chain fatty acids (LCFAs, SCFAs, and MCFAs). It dominantly occurs in mitochondria and results in acetyl-CoA (AcCoA), which could be consumed in tricarboxylic acid (TCA) cycle. For the oxidation of LCFA, it initiates from LCFA activation in cytoplasm, resulting in long-chain acyl-CoA. Subsequently, these resulting molecules are converted into long-chain acyl-carnitine by carnitine palmitoyltransferase 1 (CPT1), which is anchored on the mitochondrial outer membrane. Following its shuttling into mitochondria, long-chain acyl-carnitine experiences a chain of reactions to support FAO. Apparently, the transportation of LCFA from cytoplasm into mitochondria is a prerequisite for FAO. CPT1, the rate-limiting enzyme controlling this key step, is thus recognized as a determinant for FAO. In contrast, SCFAs and MCFAs can diffuse across mitochondrial membrane and drive FAO in a CPT1-independent manner (17). Nevertheless, extensive investigations have suggested an important role for CPT1 in iTreg differentiation (12, 13, 15, 16).In recent years, butyrate, a specific type of SCFA produced from fiber-rich diets through microbial fermentation, was shown to play a critical role in the maintenance of intestinal homeostasis and was therefore recognized as an effective ingredient from food (18–24). By modulating distinct types of immune cells, including dendritic cells (18, 19), macrophages (20), and B and T cells (21–24), butyrate contributes to the orchestration of the delicate balance in intestinal immune system. Elegant investigations have elucidated that butyrate is able to facilitate iTreg differentiation by up-regulating Foxp3 expression as a histone deacetylase (HDAC) inhibitor (22, 23). Meanwhile, butyrate, as metabolic fuel and energy source, could also support FAO in colonic epithelial cells (25). However, whether butyrate could regulate FAO to promote iTreg differentiation is unclear.In this study, we found that increased FAO contributed to enhanced iTreg cell differentiation in response to butyrate. Butyrate was processed, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which played a critical role in the control of FAO by targeting CPT1A. We found that BCoA competed with malonyl-CoA (MCoA), the best-known metabolic intermediate inhibiting CPT1A, to unleash CPT1A activity for FAO and thereby iTreg differentiation. Inhibition of ACSS2 to block BCoA generation compromised butyrate-mediated iTreg generation as well as mitigation of mouse colitis. Collectively, we depicted a previously unappreciated mechanism, namely, the butyrate–BCoA–CPT1A regulatory axis, for iTreg differentiation.     

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against β-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.

Two of the most widely studied proteins that regulate T cell activation are cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1). Inhibiting them can unleash regulatory controls of T cell activation, allowing the T cells to display their full functional potential. CTLA-4 and PD-1 are extensively studied in the cancer field, where their inhibitors—monoclonal antibodies—are used to treat cancer patients. Despite achieving success clinically, the checkpoint blockade immunotherapy can result in immune-related adverse events that frequently include endocrine autoimmune diseases (1), among them type 1 diabetes (T1D) (2–4). A recent comprehensive review summarized the clinical features of T1D induced by the immune checkpoint inhibition (4).To better understand how PD-1 is regulating T1D, we examine here the nonobese diabetic (NOD) mouse model for changes in various cellular components following PD-1 blockade. PD-1 is synthesized de novo in activated T cells mediated by T cell receptor (TCR) signaling (5–7). Whereas PD-1 expression is rapidly up-regulated after antigen stimulation of naïve T cells, sustained TCR stimulation results in substantially higher expression of PD-1 and the establishment of T cell exhaustion in the examples of chronic viral infection and cancers (8–13). NOD mice with a gene knockout of PD-1 or treated with PD-1 blocking antibody develop accelerated autoimmune diabetes shown in a number of studies (14–21). Both autoreactive CD4 and CD8 T cells can respond to PD-1/PD-L1 blockade and contribute to the acute diabetes development (17–19). Central in this process is PD-L1 (the ligand of PD-1) expressed by the islet parenchymal cells (20, 21), which limits the T cell function in the islets and protects against autoimmune diabetes.In this report we examine changes in various cellular components following PD-1 blockade by single-cell RNA sequencing (scRNA-seq) and identify a previously unexplored islet macrophage population derived from monocytes with high proinflammatory activity. In the islets of anti–PD-1–treated mice, the infiltration by monocyte-derived macrophage (MoMac) was under the influence of CD4 and particularly CD8 T cells. The CD8 T cells largely comprised the precursor exhausted T (T_PEX) cells that were activated and differentiated to produce abundant IFN-γ in response to PD-1 blockade, which in turn activated the infiltrated MoMac to promote diabetes progression. The anti–PD-1 induced development of acute diabetes was reduced by restricting the infiltration and function of such MoMac in islets. Our study establishes that the myeloid cell compartment is an indispensable component of PD-1 regulation in autoimmune diabetes. Our study provides a cellular target, the MoMac, that may minimize the adverse effects of checkpoint blockade immunotherapy.     

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell–mediated destruction of insulin-producing β-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Type 1 diabetes (T1D) is a prototypical organ-specific autoimmune disease that develops in stages (1, 2). The stages are marked by the presence of islet autoantibodies directed against insulin and other β-cell proteins, followed by impaired glucose tolerance, and finally clinical diabetes marked by hyperglycemia and the need for insulin treatment (3). The T1D disease course provides a defined preclinical period and the ability to measure immune responses prior to clinical symptoms.Self-reactive T cells target pancreatic β-cells in both murine models of spontaneous autoimmune diabetes and human T1D (4), with a number of antigens implicated as T-cell epitopes (5, 6). Recently, posttranslationally modified (PTM) epitopes have been characterized as novel autoantigens that may lead to a break in tolerance, thus resulting in T-cell–mediated immunity to pancreatic islets. PTM of antigens is well-described in autoimmune diseases, such as celiac disease (gluten sensitivity), in which tissue transglutaminase mediates deamidation of glutamine to glutamic acid within gliadin to create immunogenic CD4 T-cell epitopes (7–10). In rheumatoid arthritis, citrullinated peptides form epitopes from cartilage proteins that both elicit antibody responses and activate CD4 T cells (11). Similarly, a novel class of epitopes within T1D are hybrid insulin peptides (HIPs) that are formed within lysozymes of β-cells through a covalent bond between an insulin peptide fragment and another β-cell peptide, thereby generating a neo-epitope (12, 13).Recent studies provide strong evidence for the role of HIPs in the development of diabetes in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes (12, 14–16). Notably, the antigen for the well-studied “diabetogenic” BDC2.5 T-cell clone and transgenic mouse model is a HIP formed between a peptide fusion of C-peptide and a cleavage product of chromogranin A, termed WE14 (12, 17, 18). C-peptide is cleaved from the A and B chains of insulin prior to secretion from the β-cell. In the NOD mouse, HIP-reactive CD4 T cells have a proinflammatory phenotype, can be detected prior to the onset of diabetes, and their frequency increases as the disease progresses (15). Another CD4 T-cell epitope critical for NOD diabetes development is a fragment of the insulin B chain, consisting of amino acids 9 to 23 (B:9–23) (19–21). A strongly stimulating T-cell epitope is very likely a HIP consisting of a fragment of this insulin B-chain peptide with a portion of C-peptide fused to the C-terminal end (22). HIP-reactive CD4 T cells have also been studied in the context of human T1D, with multiple CD4 T-cell clones and lines grown from the residual pancreatic islets of T1D organ donors subsequently responding to these neo-epitopes (12, 23, 24). A number of HIP-reactive T cells have also been measured from the peripheral blood in newly diagnosed T1D patients (25–30); however, the timing of when these T cells appear in the disease course and whether these responses directed at PTM peptides precede those toward native antigens remains to be addressed. We hypothesized that HIP T-cell responses precede clinical diabetes development and are more robust than responses to native insulin peptides.In this study, we longitudinally collected peripheral blood mononuclear cells (PBMCs) from genetically at-risk individuals and measured reactivity to a panel of HIPs and native antigens using sensitive enzyme-linked immunospot (ELISPOT) assays, which have previously been used to identify CD4 T-cell responses in T1D (31). We show that PBMCs respond to native insulin peptides, but the cells respond more robustly to specific HIPs, including the insulin B chain B:9–23 HIP (B22E) and two C-peptide–derived HIPs (C-peptide/islet amyloid polypeptide-2 [C:IAPP-2] and C:A chain). We demonstrate that T-cell responses fluctuate between pro- and antiinflammatory during the preclinical phase prior to T1D development. Interestingly, individuals who progressed to clinical disease or who seroconverted to islet autoantibody positivity during the course of the study had a distinct polarization toward proinflammatory responses to specific HIPs. Remarkably, the T-cell response to the C:IAPP-2 HIP correlated with worsening measures of blood glucose. Overall, the data support a pathogenic role for PTM epitopes in the preclinical stage of T1D, and the fluctuating nature of the T-cell responses has implications for timing therapies to prevent T1D and potentially other autoimmune disorders.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed “Fab-arm exchange” in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

Recently, a growing class of antibody-mediated autoimmune diseases characterized by predominant pathogenic immunoglobulin (Ig) G4 responses has been described (1–4). IgG4 is a peculiar antibody with unique characteristics. It is, for example, unable to activate complement and has a low affinity for Fcγ receptors on immune cells (5, 6). It is therefore considered antiinflammatory, and the pathogenicity of IgG4 autoantibodies is sometimes questioned. IgG4 molecules furthermore have the unique ability to stochastically exchange half-molecules with other IgG4s in a dynamic process called “Fab-arm exchange” (7). This is an efficient process resulting in the vast majority of IgG4 molecules in circulation being bispecific and functionally monovalent for each antigen recognized. Whether the unique functional characteristics of IgG4 (like Fab-arm exchange) influence their pathogenicity in IgG4 autoimmune diseases is not known.Myasthenia gravis (MG) with antibodies against muscle-specific kinase (MuSK) is one of the first recognized IgG4-mediated autoimmune diseases. MuSK autoantibodies are predominantly of the IgG4 subclass, although anti-MuSK IgG1 and IgG3 may be present concurrently at lower titers (8). Anti-MuSK IgG4s induce MG in a dose-dependent manner both in patients and in mice (9, 10). MuSK, a receptor tyrosine kinase, has a crucial role in establishing and maintaining neuromuscular junctions (NMJs) by orchestrating postsynaptic acetylcholine receptor (AChR) clustering, which is critical for neurotransmission (11). Most MuSK autoantibodies bind the extracellular N-terminal Ig-like 1 domain and thereby block the activation of MuSK by low-density lipoprotein receptor-related protein 4 (Lrp4) and agrin (8, 12–16). This eventually leads to disassembly of densely packed AChRs in the NMJ, failure of neurotransmission, and, consequently, muscle weakness (10, 17–20). In vitro characterization of monoclonal antibodies derived from MuSK MG patients furthermore suggests that the valency of MuSK antibodies determines their effects on MuSK signaling (21–23). Monovalent Fab fragments recapitulate the inhibitory effects of patient-purified IgG4 on MuSK signaling in vitro (12, 13, 21, 23). Surprisingly, monospecific bivalent MuSK antibodies acted oppositely, as (partial) agonists (21). To investigate whether IgG4 predominance is critical for disease development in IgG4-mediated autoimmunity and study the role of Fab-arm exchange and autoantibody valency, we generated stable bispecific functionally monovalent MuSK antibodies and their monospecific bivalent equivalents and assessed their pathogenicity in NOD/SCID mice.     

Pericytes regulate vascular immune homeostasis in the CNS

Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood–brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfb^ret/ret), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfb^ret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti–ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfb^ret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfb^ret/ret;2D2^tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder.

Central nervous system (CNS) vasculature possesses specific features collectively referred to as the blood–brain barrier (BBB), which localizes to endothelial cells. The BBB ensures the delivery of essential nutrients while preventing the entry of xenobiotics into the brain. In addition, brain endothelial cells restrict the invasion of leukocytes into the brain parenchyma, thus contributing to the immune privilege of the CNS. BBB function is induced by neural tissue and established by all cell types constituting the neurovascular unit (NVU). Pericytes and mural cells residing on the abluminal side of capillaries and postcapillary venules regulate several features of the BBB (1, 2). Studies on Pdgfb and Pdgfrb mouse mutants, which exhibit variable pericyte loss, have demonstrated that pericytes negatively regulate endothelial transcytosis, which, if not suppressed, leads to increased BBB permeability to plasma proteins (1, 2). In addition, pericyte-deficient vessels show abnormal astrocyte end-feet polarization (1). Thus, pericytes regulate several characteristics of the brain vasculature during development and in the adult organism (1, 2). Whether the nonpermissive properties of the brain vasculature to leukocyte trafficking in the adult organism are regulated by pericytes has not been addressed. Interestingly, increasing evidence points to the role of pericytes in leukocyte extravasation in peripheral organs such as the skin and the striated muscle and in tumors (3–5).Increased vascular permeability to plasma proteins and immune cells accompanies neurological disorders such as multiple sclerosis (MS), stroke, and Alzheimer’s disease (reviewed in refs. 6–8). In MS, a chronic inflammatory and degenerative neurological disorder (9), autoreactive lymphocytes infiltrate the CNS parenchyma, leading to focal inflammatory infiltrates, demyelination, axonal damage, and neurodegeneration. These infiltrating immune cells could induce vascular dysfunction, including permeability to plasma proteins such as fibrinogen (10–12). On the contrary, disruption of the BBB has been shown to precede the infiltration of inflammatory cells and the formation of demyelinating lesions in MS patients (13). Therefore, it is important to understand roles of different cell types and alterations in cell–cell communication at the NVU, which may facilitate entry of autoimmune T cells as well as the anatomical localization of lesions. However, knowledge about how pericytes contribute to the development of the disease is still limited.This prompted us to investigate whether pericytes, which regulate several aspects of the BBB phenotype of endothelial cells, also regulate immune cell trafficking into the CNS during homeostasis and neuroinflammation. We show that pericytes play a crucial role in the regulation of BBB features related to the restricted leukocyte trafficking into the CNS parenchyma, both under physiological and pathophysiological conditions. We show that the permissiveness to leukocyte trafficking into the CNS inversely correlates with the vessel pericyte coverage, suggesting that vascular inflammation of the CNS due to alterations in the cellular composition of the NVU can direct the spatial distribution of neuroinflammation.     

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Cytokines are immunomodulatory proteins that have been considered for pharmaceutical applications in the treatment of cancer patients (1–3) and other types of disease (2). There is a growing interest in the use of engineered cytokine products as anticancer drugs, capable of boosting the action of T cells and natural killer (NK) cells against tumors (3, 4), alone or in combination with immune checkpoint inhibitors (3, 5–7).Recombinant cytokine products on the market include interleukin-2 (IL-2) (Proleukin) (8, 9), IL-11 (Neumega) (10, 11), tumor necrosis factor (TNF; Beromun) (12), interferon (IFN)-α (Roferon A, Intron A) (13, 14), IFN-β (Avonex, Rebif, Betaseron) (15, 16), IFN-γ (Actimmune) (17), granulocyte colony-stimulating factor (Neupogen) (18), and granulocyte macrophage colony-stimulating factor (Leukine) (19, 20). The recommended dose is typically very low (often <1 mg/d) (21–23), as cytokines may exert biological activity in the subnanomolar concentration range (24). Various strategies have been proposed to develop cytokine products with improved therapeutic index. Protein PEGylation or Fc fusions may lead to prolonged circulation time in the bloodstream, allowing the administration of low doses of active payload (25, 26). In some implementations, cleavable polyethylene glycol polymers may be considered, yielding prodrugs that regain activity at later time points (27). Alternatively, tumor-homing antibody fusions have been developed, since the preferential concentration of cytokine payloads at the tumor site has been shown in preclinical models to potentiate therapeutic activity, helping spare normal tissues (28–34). Various antibody-cytokine fusions are currently being investigated in clinical trials for the treatment of cancer and of chronic inflammatory conditions (reviewed in refs. 2, 33, 35–37).Antibody-cytokine fusions display biological activity immediately after injection into patients, which may lead to unwanted toxicity and prevent escalation to therapeutically active dosage regimens (9, 22, 38). In the case of proinflammatory payloads (e.g., IL-2, IL-12, TNF-α), common side effects include hypotension, nausea, and vomiting, as well as flu-like symptoms (24, 39–42). These side effects typically disappear when the cytokine concentration drops below a critical threshold, thus providing a rationale for slow-infusion administration procedures (43). It would be highly desirable to generate antibody-cytokine fusion proteins with excellent tumor-targeting properties and with “activity on demand”— biological activity that is conditionally gained on antigen binding at the site of disease, helping spare normal tissues.Here we describe a fusion protein consisting of the F8 antibody specific to the alternatively spliced extra domain A (EDA) of fibronectin (44, 45) and of murine 4-1BBL, which did not exhibit cytokine activity in solution but could regain potent biological activity on antigen binding. The antigen (EDA+ fibronectin) is conserved from mouse to man (46), is virtually undetectable in normal adult tissues (with the exception of the placenta, endometrium, and some vessels in the ovaries), but is expressed in the majority of human malignancies (44, 45, 47, 48). 4-1BBL, a member of the TNF superfamily (49), is expressed on antigen-presenting cells (50, 51) and binds to its receptor, 4-1BB, which is up-regulated on activated cytotoxic T cells (52), activated dendritic cells (52), activated NK and NKT cells (53), and regulatory T cells (54). Signaling through 4-1BB on cytotoxic T cells protects them from activation-induced cell death and skews the cells toward a more memory-like phenotype (55, 56).We engineered nine formats of the F8-4-1BBL fusion protein, one of which exhibited superior performance in quantitative biodistribution studies and conditional gain of cytokine activity on antigen binding. The antigen-dependent reconstitution of the biological activity of the immunostimulatory payload represents an example of an antibody fusion protein with “activity on demand.” The fusion protein was potently active against different types of cancer without apparent toxicity at the doses used. The EDA of fibronectin is a particularly attractive antigen for cancer therapy in view of its high selectivity, stability, and abundant expression in most tumor types (44, 45, 47, 48).