Neuroinflammation in neuronopathic Gaucher disease: Role of microglia and NK cells, biomarkers, and response to substrate reduction therapy.

Background: Neuronopathic Gaucher disease (nGD) is a rare neurodegenerative disorder caused by biallelic mutations in GBA and buildup of glycosphingolipids in lysosomes. Neuronal injury and cell death are prominent pathological features; however, the role of GBA in individual cell types and involvement of microglia, blood-derived macrophages, and immune infiltrates in nGD pathophysiology remains enigmatic. Methods: Here, using single-cell resolution of mouse nGD brains, lipidomics, and newly generated biomarkers, we found induction of neuroinflammation pathways involving microglia, NK cells, astrocytes, and neurons. Results: Targeted rescue of Gba in microglia and neurons, respectively, in Gba-deficient, nGD mice reversed the buildup of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph), concomitant with amelioration of neuroinflammation, reduced serum neurofilament light chain (Nf-L), and improved survival. Serum GlcSph concentration was correlated with serum Nf-L and ApoE in nGD mouse models as well as in GD patients. Gba rescue in microglia/macrophage compartment prolonged survival, which was further enhanced upon treatment with brain-permeant inhibitor of glucosylceramide synthase, effects mediated via improved glycosphingolipid homeostasis, and reversal of neuroinflammation involving activation of microglia, brain macrophages, and NK cells. Conclusions: Together, our study delineates individual cellular effects of Gba deficiency in nGD brains, highlighting the central role of neuroinflammation driven by microglia activation. Brain-permeant small-molecule inhibitor of glucosylceramide synthase reduced the accumulation of bioactive glycosphingolipids, concomitant with amelioration of neuroinflammation involving microglia, NK cells, astrocytes, and neurons. Our findings advance nGD disease biology whilst identifying compelling biomarkers of nGD to improve patient management, enrich clinical trials, and illuminate therapeutic targets. Funding: Research grant from Sanofi; other support includes R01NS110354, Yale Liver Center P30DK034989, pilot project grant.

Antigen-mediated regulation in monoclonal gammopathies and myeloma.

A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies. Here, we show that recombinant Ig (rIg) cloned from sort-purified single tumor cells from lipid-reactive sporadic and GD-associated gammopathy specifically bound lysolipids. Liposome sedimentation and binding assays confirmed specific interaction of lipid-reactive monoclonal Ig with lysolipids. The clonal nature of lysolipid-binding Ig was validated by protein sequencing. Gene expression profiling and cytogenetic analyses from 2 patient cohorts showed enrichment of nonhyperdiploid tumors in lipid-reactive patients. In vivo antigen-mediated stimulation led to an increase in clonal Ig and plasma cells (PCs) in GD gammopathy and also reactivated previously suppressed antigenically related nonclonal PCs. These data support a model wherein antigenic stimulation mediates an initial polyclonal phase, followed by evolution of monoclonal tumors enriched in nonhyperdiploid genomes, responsive to underlying antigen. Targeting underlying antigens may therefore prevent clinical MM.

Clinical and Serologic Responses After a Two-dose Series of High-dose Influenza Vaccine in Plasma Cell Disorders: A Prospective, Single-arm Trial.

BACKGROUND: Patients with multiple myeloma (MM) and other plasma cell disorders are highly susceptible to influenza infections, which are major causes of morbidity in this population, despite the routine administration of a seasonal influenza vaccination. Existing data are limited by small and retrospective studies, which suggest poor seroprotection rates of < 20% after standard influenza vaccination in patients with MM. PATIENTS AND METHODS: Patients with plasma cell dyscrasia (n = 51) were treated with a 2-dose series of high-dose inactivated trivalent influenza vaccine during the 2014 to 2015 influenza season. Laboratory-confirmed influenza infections were identified through seasonal surveillance, sera were collected for influenza hemagglutination antibody inhibition (HAI) titer assays, and logistic regression models were used to identify the clinical correlates to the HAI serologic responses. RESULTS: Influenza vaccine was well tolerated, without any vaccine-related grade ≥ 2 adverse events. Only 3 patients (6%) experienced laboratory-confirmed influenza. The rates of HAI seroprotection against all 3 vaccine strains (A/California/7/2009 [H1N1] pdm09-like virus; A/Texas/50/2012 [H3N2]-like virus; and a B/Massachusetts/2/2012-like virus) increased from 4% at baseline to 49% and 65% after 1 and 2 doses, respectively. The risk factors associated with a lower likelihood of HAI serologic response included plasma cell disorder requiring therapy, less than a partial response found on disease response assessment, and active conventional chemotherapy. Alternatively, active therapy with an immunomodulatory drug alone or with a proteasome inhibitor was associated with a greater likelihood of an HAI serologic response. CONCLUSION: These data have demonstrated that, in contrast to the historically poor results with standard influenza vaccination, this novel high-dose booster vaccination strategy leads to high rates of seroprotection. Randomized controlled studies are needed to compare this novel strategy to the standard vaccination strategy.

Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells.

Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.

ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells.

Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.

Clonal Immunoglobulin against Lysolipids in the Origin of Myeloma.

Antigen-driven selection has been implicated in the pathogenesis of monoclonal gammopathies. Patients with Gaucher's disease have an increased risk of monoclonal gammopathies. Here we show that the clonal immunoglobulin in patients with Gaucher's disease and in mouse models of Gaucher's disease-associated gammopathy is reactive against lyso-glucosylceramide (LGL1), which is markedly elevated in these patients and mice. Clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC). Substrate reduction ameliorates Gaucher's disease-associated gammopathy in mice. Thus, long-term immune activation by lysolipids may underlie both Gaucher's disease-associated gammopathies and some sporadic monoclonal gammopathies.

Conditional overexpression of TGFß1 promotes pulmonary inflammation, apoptosis and mortality via TGFßR2 in the developing mouse lung.

BACKGROUND: Earlier studies have reported that transforming growth factor beta 1(TGFß1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFß1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFß1 signaling via TGFßR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI. METHODS: We utilized lung epithelial cell-specific TGFß1 overexpressing transgenic and TGFßR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed. RESULTS: Our data reveals that increased TGFß1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers. CONCLUSIONS: Our study has demonstrated the potential role of inhibition of TGFß1 signaling via TGFßR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that ß-glucosylceramide 22:0 (ßGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human ßGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, ßGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human ßGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.

Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinct immunologic changes in vivo.

Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to remarkable antitumor effects. Although both PD-1 and CTLA-4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. To better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA-4, PD-1, or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely nonoverlapping changes in coding genes, including alternatively spliced transcripts and noncoding RNAs. Pathway analysis revealed that CTLA-4 blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, whereas PD-1 blockade instead leads to changes in genes implicated in cytolysis and NK cell function. Combination blockade leads to nonoverlapping changes in gene expression, including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, soluble IL-2R, and IL-1α. Importantly, PD-1 receptor occupancy following anti-PD-1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that, despite shared property of checkpoint blockade, Abs against PD-1, CTLA-4 alone, or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agents in patients will support rational development of immune-based combinations against cancer.

Amyloid fibrils enhance transport of metal nanoparticles in living cells and induced cytotoxicity.

Amyloid protein fibrils occur in vivo as pathological agents, in the case of neurodegenerative diseases, or as functional amyloids, when playing biologically vital roles. Here we show how amyloid fibrils generated from a food protein, ß-lactoglobulin, can be used as nanoreactors for the synthesis of metal nanoparticles and demonstrate that the resulting hybrids can play a central role in the internalization of nanoparticles into living cells, with up to 3-fold-enhanced transport properties over pristine nanoparticles. We conjugate gold, silver, and palladium nanoparticles onto amyloid fibrils by chemical reduction, and we study their effect on dendritic and MCF7 breast cancer cells. Transmission electron microscopy indicates localization of nanoparticles inside vesicles of the cells. Flow cytometry reveals that silver nanoparticle-amyloid hybrids are cytotoxic, while gold and palladium nanoparticle-amyloid hybrids produce no notable effect on cell viability and activation status.

Dendritic cell homeostasis is maintained by nonhematopoietic and T-cell-produced Flt3-ligand in steady state and during immune responses.

Lymphoid-tissue dendritic cells (DCs) are short-lived and need to be continuously replenished from bone marrow-derived DC progenitor cells. Fms-related tyrosine kinase 3 is expressed during cellular development from hematopoietic progenitors to lymphoid-tissue DCs. Fms-related tyrosine kinase 3 ligand (Flt3L) is an essential, nonredundant cytokine for DC progenitor to lymphoid tissue DC differentiation and maintenance. However, which cells contribute to Flt3L production and how Flt3L cytokine levels are regulated in steady state and during immune reactions remains to be determined. Here we demonstrate that besides nonhematopoietic cells, WT T cells produce Flt3L and contribute to the generation of both classical DCs (cDCs) and plasmacytoid DCs in Flt3L(-/-) mice. Upon stimulation in vitro, CD4(+) T cells produce more Flt3L than CD8(+) T cells. Moreover, in vivo stimulation of naïve OT-II CD4(+) T cells with OVA leads to increase of pre-cDCs and cDCs in draining lymph nodes of Flt3L(-/-) mice in a partially Flt3L-dependent manner. Thus, Flt3L-mediated lymphoid tissue DC homeostasis is regulated by steady-state T cells as well as by proliferative T cells, fostering local development of lymphoid organ resident DCs.

Nitric oxide inhibits interleukin-12 p40 through p38 MAPK-mediated regulation of calmodulin and c-rel.

In activated macrophages, the rel/NF-kappaB transcription factors are known to play important roles in interleukin-12 (IL-12) p40 regulation by nitric oxide (NO). However, the relative contributions of these factors are not well understood. Here, we describe a dominant role for c-rel involving p38 mitogen-activated protein kinase (p38 MAPK) and calmodulin (CaM) protein in NO-mediated IL-12 p40 inhibition in activated macrophages. Inhibition of NO production by aminoguanidine increased, whereas sodium nitroprusside (SNP; an exogenous NO generator) reduced, nuclear c-rel levels in LPS + IFN-gamma-activated RAW 264.7 macrophages. Overexpression of c-rel but not p65 NF-kappaB increased IL-12 p40 during NO treatment. The p38 MAPK phosphorylation is increased by NO, and inhibition of p38 MAPK in SNP-treated macrophages by SB203580 or transient expression of a dominant-negative mutant of p38 MAPK upregulated both nuclear c-rel and IL-12 p40 levels, indicating that NO targeted the p38 MAPK pathway to inhibit c-rel and IL-12 p40. Cytoplasmic CaM level was increased by NO, and SB203580 decreased the CaM level in NO-exposed macrophages. Inhibition of CaM activity by trifluoperazine rescued the inhibitory effect of NO on c-rel and IL-12 p40. Our findings indicate that c-rel plays an important role in NO-mediated inhibition of IL-12 p40 and is regulated by p38 MAPK through CaM protein.

Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein.

Although the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H2O2), a major component of ROSs, inhibits interleukin-12 (IL-12) p40 and IL-12 p70 induction in murine macrophages and catalase pretreatment prevents H2O2-mediated down-regulation of IL-12. Endogenous accumulation of H2O2/ROSs in macrophages treated with alloxan resulted in IL-12 p40 inhibition. Although nuclear expression of both p50 and p65 NF-kappaB increased on H2O2 exposure, nuclear c-rel level was inhibited. Overexpression of c-rel restored IL-12 p40 on stimulation with lipopolysaccharide plus IFN-gamma during H2O2 treatment. H2O2 did not inhibit c-rel induction in cytosol; however, it prevented the transport of c-rel from cytosol to the nucleus. H2O2 activated calmodulin (CaM) protein in the cytosol, which subsequently sequestered c-rel in the cytosol preventing its transport to the nucleus. The CaM inhibitor trifIuoperazine increased both nuclear c-rel and IL-12 p40 levels in H2O2-treated macrophages, emphasizing a role of CaM in these processes. H2O2/ROSs thus down-regulate IL-12 induction in macrophages by a novel pathway inhibiting c-rel translocation to the nucleus through activation of CaM protein.

Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor.

Interleukin-10 (IL-10) is known to inhibit IL-12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor-kappaB (NF-kappaB). We demonstrate that the c-rel transcription factor also plays a major role in IL-10-mediated IL-12 suppression. Treatment of macrophages with recombinant IL-10 inhibited nuclear c-rel levels, whereas addition of neutralizing anti-IL-10 antibody up-regulated both nuclear c-rel levels and IL-12 production by macrophages. Decreased nuclear c-rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IkappaBalpha) in the cytoplasm, indicating that IL-10 prevents degradation of IkappaBalpha and the subsequent translocation of c-rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c-rel at a concentration of 10 nm, when used with anti-IL-10 antibody, resulted in reduced expression of IL-12. In a complementary experiment, in vitro transient expression of p65 NF-kappaB could not rescue the inhibitory effect of IL-10 on IL-12 production, suggesting that NF-kappaB alone was not sufficient to restore IL-12 production during IL-10 treatment. However, over-expression of c-rel resulted in IL-12 restoration upon stimulation with lipopolysaccharide plus interferon-gamma during IL-10 treatment. Our studies highlight the involvement of c-rel in IL-10-mediated IL-12 regulation.