TLR9/MyD88 signaling is required for class switching to pathogenic IgG2a and 2b autoantibodies in SLE

Loss of tolerance in systemic lupus erythematosus (SLE) leads to the generation of autoantibodies, which accumulate in end-organs where they induce disease. Here we show that immunoglobulin (Ig)G2a and 2b autoantibodies are the pathogenic isotypes by recruiting FcγRIV expressing macrophages. Class switching, but not development, of IgM anti-self B cells to these pathogenic subclasses requires the innate immune receptor Toll-like receptor (TLR)9 and MyD88 signaling. In their absence, switching of autoreactive B cells to the IgG2a and 2b subclasses is blocked, resulting in reduced pathology and mortality. In contrast, switching of anti-self B cells to IgG1 is not perturbed and generation of nonautoreactive IgG2a and 2b antibodies is not impaired in TLR9-deficient mice. Thus, the TLR9 pathway is a potential target for therapeutic intervention in SLE

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials

LAG-3-deficient mice produce higher amounts of cytokine following mercury treatment.

<p>Wild-type and <i>Lag-3^−/−</i> mice were either given a challenge dose of HgCl₂ (30 µg/mouse s.c. on days 0, 2 and 4) or were left untreated. Splenocytes were harvested on day 8 and then stimulated in vitro with plate bound anti-CD3 and anti-CD28 antibodies. Supernatant were collected after 72 hrs to determine the levels of cytokine by ELISA (n = 3 or 4). Two way ANOVA was used to determine the statistical significance between the groups where *indicates <i>p</i><0.05 and ***indicates <i>p</i><0.0001.</p

Cutting Edge: DNA Sensing via the STING Adaptor in Myeloid Dendritic Cells Induces Potent Tolerogenic Responses

Cytosolic DNA sensing via the STING adaptor incites autoimmunity by inducing type I IFN (IFNαβ). Here we show that DNA is also sensed via STING to suppress immunity by inducing indoleamine 2,3 dioxygenase (IDO). STING gene ablation abolished IFNαβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs and myeloid cells ingested DNPs but CD11b(+) DCs were the only cells to express IFNβ, while CD11b(+) non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells (Tregs), and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate (c-diGMP) treatment to activate STING induced selective IFNβ expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity amongst physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA

Anti-LAG-3 monoclonal antibody breaks established tolerance against mercury-induced autoimmunity.

<p>A.SW mice were tolerized to Hg and 7 days later they received a challenge dose of Hg in conjunction with anti-LAG-3 or control antibody as depicted in the Figure. Serum IgG1 and IgE levels were measured by ELISA and autoantibody titers were determined by IF assay (n = 5). The IgG and IgE concentrations depicted at wk -1 represent the baseline levels of antibodies in serum of untreated mice and no autoantibodies were detectable in untreated mice. To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p