Reversal of global CD4+ subset dysfunction is associated with spontaneous clinical resolution of pulmonary sarcoidosis.

Sarcoidosis pathogenesis is characterized by peripheral anergy and an exaggerated, pulmonary CD4(+) Th1 response. In this study, we demonstrate that CD4(+) anergic responses to polyclonal TCR stimulation are present peripherally and within the lungs of sarcoid patients. Consistent with prior observations, spontaneous release of IL-2 was noted in sarcoidosis bronchoalveolar lavage CD4(+) T cells. However, in contrast to spontaneous hyperactive responses reported previously, the cells displayed anergic responses to polyclonal TCR stimulation. The anergic responses correlated with diminished expression of the Src kinase Lck, protein kinase C-θ, and NF-κB, key mediators of IL-2 transcription. Although T regulatory (Treg) cells were increased in sarcoid patients, Treg depletion from the CD4(+) T cell population of sarcoidosis patients did not rescue IL-2 and IFN-γ production, whereas restoration of the IL-2 signaling cascade, via protein kinase C-θ overexpression, did. Furthermore, sarcoidosis Treg cells displayed poor suppressive capacity indicating that T cell dysfunction was a global CD4(+) manifestation. Analyses of patients with spontaneous clinical resolution revealed that restoration of CD4(+) Th1 and Treg cell function was associated with resolution. Conversely, disease progression exhibited decreased Th1 cytokine secretion and proliferative capacity, and reduced Lck expression. These findings implicate normalized CD4(+) T cell function as a potential therapeutic target for sarcoidosis resolution.

Blockade of the programmed death-1 pathway restores sarcoidosis CD4(+) T-cell proliferative capacity.

RATIONALE: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. OBJECTIVES: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4(+) T-cell proliferative capacity. METHODS: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage-derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1(+) CD4(+) T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1(+) CD4(+) T cells with spontaneous clinical resolution but not with disease progression. CONCLUSIONS: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4(+) T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a viable therapeutic target to optimize clinical outcomes.

Sarcoidosis Th17 cells are ESAT-6 antigen specific but demonstrate reduced IFN-γ expression.

RATIONALE: Sarcoidosis is a granulomatous disease of unknown etiology. Many patients with sarcoidosis demonstrate antigen-specific immunity to mycobacterial virulence factors. Th-17 cells are crucial to the immune response in granulomatous inflammation, and have recently been shown to be present in greater numbers in the peripheral blood and bronchoalveolar lavage (BAL) fluid (BALF) of sarcoidosis patients than healthy controls. It is unclear whether Th-17 cells in sarcoidosis are specific for mycobacterial antigens, or whether they have similar functionality to control Th-17 cells. METHODS: Flow cytometry was used to determine the numbers of Th-17 cells present in the peripheral blood and BALF of patients with sarcoidosis, the percentage of Th-17 cells that were specific to the mycobacterial virulence factor ESAT-6, and as well as to assess IFN-γ expression in Th-17 cells following polyclonal stimulation. RESULTS: Patients with sarcoidosis had greater numbers of Th-17 cells in the peripheral blood and BALF than controls and produced significantly more extracellular IL-17A (p = 0.03 and p = 0.02, respectively). ESAT-6 specific Th-17 cells were present in both peripheral blood and BALF of sarcoidosis patients (p < 0.001 and p = 0.03, respectively). After polyclonal stimulation, Th-17 cells from sarcoidosis patients produced less IFN-γ than healthy controls. CONCLUSIONS: Patients with sarcoidosis have mycobacterial antigen-specific Th-17 cells peripherally and in sites of active sarcoidosis involvement. Despite the Th1 immunophenotype of sarcoidosis immunology, the Th-17 cells have reduced IFN-γ expression, compared to healthy controls. This reduction in immunity may contribute to sarcoidosis pathogenesis.

Development of spontaneous anergy in invariant natural killer T cells in a mouse model of dyslipidemia.

OBJECTIVE: In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells. METHODS AND RESULTS: We compared iNKT cell expansion and cytokine production in C57BL/6J (B6) and apolipoprotein E-deficient (apoE(-/-)) mice. In response to in vivo stimulation with alpha-galactosylceramide, a prototypic iNKT cell glycolipid antigen, apoE(-/-) mice showed significantly decreased splenic iNKT cell expansion at 3 days after injection, a profile associated with iNKT cell anergy due to chronic stimulation. This decrease in expansion and cytokine production was accompanied by a 2-fold increase in percentage of iNKT cells expressing the inhibitory marker programmed death-1 in apoE(-/-) mice compared with controls. However, in vivo and in vitro blockade of programmed death-1 using monoclonal antibody was not able to restore functions of iNKT cells from apoE(-/-) mice to B6 levels. iNKT cells from apoE(-/-) mice also had increased intracellular T cell receptor and Ly49 expression, a phenotype associated with previous activation. Changes in iNKT cell functions were cell autonomous, because dendritic cells from apoE(-/-) mice were able to activate B6 iNKT cells, but iNKT cells from apoE(-/-) mice were not able to respond to B6 dendritic cells. CONCLUSIONS: These data suggest that chronic dyslipidemia induces an iNKT cell phenotype that is unresponsive to further simulation by exogenous glycolipid and that sustained unresponsiveness is iNKT cell intrinsic.

The inhibitory FcγRIIb modulates the inflammatory response and influences atherosclerosis in male apoE(-/-) mice.

BACKGROUND: Atherosclerosis is widely accepted as an inflammatory disease involving both innate and adaptive immunity. B cells and/or antibodies have previously been shown to play a protective role against atherosclerosis. Aside from their ability to bind to antigens, antibodies can influence inflammatory responses by interacting with various Fcγ receptors on the surface of antigen presenting cells. Although studies in mice have determined that stimulatory Fcγ receptors contribute to atherosclerosis, the role of the inhibitory Fcγ receptor IIb (FcγRIIb) has only recently been investigated. METHODS AND RESULTS: To determine the importance of FcγRIIb in modulating the adaptive immune response to hyperlipidemia, we generated FcγRIIb-deficient mice on the apoE-deficient background (apoE/FcγRIIb(-/-)). We report that male apoE/FcγRIIb(-/-) mice develop exacerbated atherosclerosis that is independent of lipid levels, and is characterized by increased antibody titers to modified LDL and pro-inflammatory cytokines in the aorta. CONCLUSIONS: These findings suggest that antibodies against atherosclerosis-associated antigens partially protect against atherosclerosis in male apoE(-/-) mice by conveying inhibitory signals through the FcγRIIb that downregulate pro-inflammatory signaling via other immune receptors. These data are the first to describe a significant in vivo effect for FcγRIIb in modulating the cytokine response in the aorta in male apoE(-/-) mice.

Natural killer T cells and atherosclerosis: form and function meet pathogenesis.

Atherosclerosis is a chronic inflammatory disease characterized by dyslipidemia and accumulation of lipids in the arterial intima, with activation of both innate and adaptive immunity. Reciprocally, dyslipidemia associated with atherosclerosis can perturb normal immune function. Natural killer T (NKT) cells are a specialized group of immune cells that share characteristics with both conventional T cells and natural killer cells. However, unlike these cells, NKT cells recognize glycolipid antigens and produce both pro- and anti-inflammatory cytokines upon activation. Because of these unique characteristics, NKT cells have recently been ascribed a role in the regulation of immunity and inflammation, including cardiovascular disease. In addition, NKT cells represent a bridge between dyslipidemia and immune regulation. This review summarizes the current knowledge of NKT cells and discusses the interplay between dyslipidemia and the normal functions of NKT cells and how this might modulate inflammation and atherosclerosis.

Intracellular trafficking of recycling apolipoprotein E in Chinese hamster ovary cells.

We have investigated apolipoprotein E (apoE) recycling in Chinese hamster ovary (CHO) cells, a peripheral cell that does not produce lipoproteins or express apoE. Using a pulse-chase protocol in which cells were pulsed with 125I-apoE-VLDL and chased for different periods, approximately 30% of the apoE internalized during the pulse was resecreted within a 4 h chase in a relatively lipid-free state. The addition of lysosomotropic agents or brefeldin A had no effect on apoE recycling. Unlike previous results with hepatocytes and macrophages, neither apoA-I nor upregulation of ABCA1 stimulated apoE recycling. However, cyclodextrin, which extracts cholesterol from plasma membrane lipid rafts, increased recycling. Confocal studies revealed that apoE, internalized during a 1 h pulse, colocalizes with early endosomal antigen-1, Rab5, Rab11a, and lysobisphosphatidic acid but not with lysosomal-associated membrane protein-1. Colocalization of apoE and Rab11a persisted even after cells had been chased for 1 h, suggesting a pool of apoE within the endosomal recycling compartment (ERC). Our data suggest that apoE recycling in CHO cells is linked to cellular cholesterol removal via the ERC and phospholipid-containing acceptors in a pathway alternative to the ABCA1-apoA-I axis.