Publications

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, has been shown to control T cell function. We found that in vitro-activated B cells and B cells from various lupus-prone mice and patients with systemic lupus erythematosus display increased PP2A activity. To understand the contribution of PP2A to B cell function, we generated a Cd19CrePpp2r1afl/fl (flox/flox) mouse which lacks functional PP2A only in B cells. Flox/flox mice displayed reduced spontaneous germinal center formation and decreased responses to T cell-dependent and T-independent antigens, while their B cells responded poorly in vitro to stimulation with an anti-CD40 antibody or CpG in the presence of IL-4. Transcriptome and metabolome studies revealed altered nicotinamide adenine dinucleotide (NAD) and purine/pyrimidine metabolism and increased expression of purine nucleoside phosphorylase in PP2A-deficient B cells. Our results demonstrate that PP2A is required for optimal B cell function and may contribute to increased B cell activity in systemic autoimmunity.

Autoimmune connective tissue diseases arise in a stepwise fashion from asymptomatic preclinical autoimmunity. Type I interferons have a crucial role in the progression to established autoimmune diseases. The cellular source and regulation in disease initiation of these cytokines is not clear, but plasmacytoid dendritic cells have been thought to contribute to excessive type I interferon production. Here, we show that in preclinical autoimmunity and established systemic lupus erythematosus, plasmacytoid dendritic cells are not effector cells, have lost capacity for Toll-like-receptor-mediated cytokine production and do not induce T cell activation, independent of disease activity and the blood interferon signature. In addition, plasmacytoid dendritic cells have a transcriptional signature indicative of cellular stress and senescence accompanied by increased telomere erosion. In preclinical autoimmunity, we show a marked enrichment of an interferon signature in the skin without infiltrating immune cells, but with interferon-kappa production by keratinocytes. In conclusion, non-hematopoietic cellular sources, rather than plasmacytoid dendritic cells, are responsible for interferon production prior to clinical autoimmunity.

Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2-enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell-specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.

The cellular origin of CD4(-) CD8(-) (double negative, DNT) TCR-alpha/beta(+) T cells remains unknown. Available evidence indicates that they may derive from CD8(+) T cells, but most published data have been obtained using cells that bear an invariant transgenic T cell receptor that recognizes an Ag that is not present in normal mice. Here, we have used complementary fate mapping and adoptive transfer experiments to identify the cellular lineage of origin of DNT cells in wild-type mice with a polyclonal T cell repertoire. We show that TCR-alpha/beta(+) DNT cells can be traced back to CD8(+) and CD4(+) CD8(+) double positive cells in the thymus. We also demonstrate that polyclonal DNT cells generated in secondary lymphoid organs proliferate upon adoptive transfer and can regain CD8 expression in lymphopenic environment. These results demonstrate the cellular origin of DNT cells and provide a conceptual framework to understand their presence in pathological circumstances.

BACKGROUND: Activation of the complement system and complement deposition on red blood cells (RBCs) contribute to organ damage in trauma. We conducted a prospective study in subjects with traumatic injuries to determine the pattern of complement deposition on RBC and whether they are associated with clinical outcomes. METHOD: A total of 124 trauma patients and 42 healthy controls were enrolled in this prospective study. RBC and sera were collected at 0, 6, 24, and 72 h from trauma patients and healthy controls during a single draw. Presence of C4d, C3d, C5b-9, phosphorylation of band 3 and production of nitric oxide were analyzed by flow cytometry. RESULTS: RBC from trauma patients at all time points up to 24 h displayed significantly higher deposition of C4d on their RBC membrane as compared with healthy donors. Incubation of normal RBC with sera from trauma patients resulted in significant increase of C4d deposition (at 0, 6, 24, and 72 h), C5b-9 deposition (at 0 and 6 h), phosphorylation of band 3 (at 0 and 24 h), and nitric oxide production up to 24 h compared with sera from healthy subjects. Deposition of C4d and C5b-9 in patients with an Injury Severity Score (ISS) of 9 and above remained elevated up to 72 h. CONCLUSIONS: Our study demonstrates that the presence of C4d, C3d, and C5b-9 on the surface of RBC is linked to increased phosphorylation of band 3 and increased production of nitric oxide. Deposition of C4d and C5b-9 decreased faster over course of 3-day study in subjects with ISS less than 9.

How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55beta, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8+ T cells. Through the modulation of AKT, B55beta induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55beta and Hrk were both required for in vivo and in vitro contraction of activated CD8+ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory.

T cell subsets are critically involved in the development of systemic autoimmunity and organ inflammation in systemic lupus erythematosus (SLE). Each T cell subset function (such as effector, helper, memory or regulatory function) is dictated by distinct metabolic pathways requiring the availability of specific nutrients and intracellular enzymes. The activity of these enzymes or nutrient transporters influences the differentiation and function of T cells in autoimmune responses. Data are increasingly emerging on how metabolic processes control the function of various T cell subsets and how these metabolic processes are altered in SLE. Specifically, aberrant glycolysis, glutaminolysis, fatty acid and glycosphingolipid metabolism, mitochondrial hyperpolarization, oxidative stress and mTOR signalling underwrite the known function of T cell subsets in patients with SLE. A number of medications that are used in the care of patients with SLE affect cell metabolism, and the development of novel therapeutic approaches to control the activity of metabolic enzymes in T cell subsets represents a promising endeavour in the search for effective treatment of systemic autoimmune diseases.

Current technologies and available scaffold materials do not support long-term cell viability, differentiation and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. We developed a new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding. Human immortalized podocytes cultured on this platform displayed superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displayed a softer surface and an abundance of growth factors and associated molecules. More importantly it enabled podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.