the-isolation-differentiation-quantification-human-antibody-secreting

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

National Taiwan University

The cells of both the adaptive and innate immune systems express a dizzying array of receptors that transduce and integrate an enormous amount of information about the environment that allows the cells to mount effective immune responses. Over the past several years, significant advances have been made in elucidating the molecular details of signal cascades initiated by the engagement of immune cell receptors by their ligands. Recent evidence indicates that immune receptors and components of their signaling cascades are spatially organized and that this spatial organization plays a central role in the initiation and regulation of signaling. A key organizing element for signaling receptors appears to be cholesterol- and sphingolipid-rich plasma membrane microdomains termed lipid rafts. Research into the molecular basis of the spatial segregation and organization of signaling receptors provided by rafts is adding fundamentally to our understanding of the initiation and prolongation of signals in the immune system.

Location is everything: lipid rafts and immune cell signaling.

Recent advances in cell biology have provided evidence that the plasma membrane is not a homogeneous lipid bilayer but rather contains within it sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, which serve as platforms for both receptor signaling and trafficking. In B lymphocytes lipid rafts appear to play a key role in the initiation of B-cell antigen receptor (BCR) signaling. Current methods to isolate lipid rafts rely on the relative detergent insolubility of lipid rafts as compared to the nonraft, glycerophospholipid bilayer. Here a method to isolate and characterize lipid rafts from B lymphocytes is described. Particular emphasis is given to the potential artifacts inherent in current procedures that rely on detergents to isolate lipid rafts and alternative technologies that may circumvent these.

Isolation of lipid rafts from B lymphocytes.

The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.

The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway.

The FcgammaRIIB is a potent regulator of BCR signaling and as such plays a decisive role in controlling autoimmunity. The use of advanced imaging technologies has provided evidence that the earliest events in Ag-induced BCR signaling include the clustering of the BCR, the selective and transient association of the clustered BCR with raft lipids, and the concentration of BCR clusters in an immune synapse. That lipid rafts play a role in FcgammaRIIB's regulation of BCR signaling was suggested by recent studies showing that a lupus-associated loss of function mutation resulted in the receptor's exclusion from lipid rafts and the failure to regulate BCR signaling. In this study, we provide evidence from both biochemical analyses and fluorescence resonance energy transfer in conjunction with both confocal and total internal reflection microscopy in living cells that the FcgammaRIIB, when coligated with the BCR, associates with lipid rafts and functions both to destabilize the association of the BCR with raft lipids and to block the subsequent formation of the B cell's immune synapse. These results define new early targets of FcgammaRIIB inhibitory activity in the Ag-induced B cell activation pathway.

Live cell imaging reveals that the inhibitory FcgammaRIIB destabilizes B cell receptor membrane-lipid interactions and blocks immune synapse formation.

AMPK was shown to negatively regulate FcεRI activation, and FcεR-mediated Fyn activation can counteract the LKB1/AMPK axis in mast cells. However, the relationship between the major Src family kinase Lyn and AMPK remains poorly defined. Here, we investigate the molecular mechanism for AMPK inhibition by FcεRI-Lyn signaling in rat RBL-2H3 cells. We found that FcεRI activation could rapidly inhibit AMPK activation through increased AMPK phosphorylation at the inhibitory Ser485/491 residues without a change at the activating Th172 residue, and this was accompanied by a reduction of ACC phosphorylation. Using specific inhibitors and gene silencing, we found that such AMPK inhibition involved a signaling cascade through Lyn-Syk-Akt. When AMPK was activated by AICAR, A769662 and metformin, FcεRI-mediated Syk, ERK, JNK and p38 activation, and TNFα release were all inhibited. Consistently, AMPK inhibition by compound C increased FcεRI-mediated Lyn activation. Moreover, AMPK activation dominantly impaired IgE-induced recruitment of signal proteins to the FcεRI by blocking the formation of FcεRIβ-Lyn-Syk, FcεRIγ-Lyn-Syk, and AMPK-FcεRIβ complexes. In vitro kinase assay further revealed the ability of AMPKα2 to phosphorylate FcεRIβ in the complex. In vivo, AMPK activation by metformin could readily reduce vascular permeability and ear swelling in a mouse model of passive cutaneous anaphylaxis mediated by IgE. In summary, our findings demonstrate that IgE-mediated FcεRI activation results in AMPK inhibition through activation of Lyn-Syk-Akt pathway, and as such FcεRI receptor can efficiently propagate Lyn-mediated allergic signaling and response. These results provide important insights into the use of AMPK activators for the treatment of allergic diseases.

Inhibition of AMPK through Lyn-Syk-Akt enhances FcεRI signal pathways for allergic response.

The inhibitory Fc receptor, FcγRIIB, has emerged as a key negative regulator of B cell activation and as such is predicted to play an essential role in controlling antibody-mediated autoimmune diseases in humans. Recent studies have shown that crosslinking the FcγRIIB independently of the B-cell receptor (BCR) results in apoptosis in both mouse and chicken B cells. However, the human B cell subpopulations that are susceptible to BCR-independent, FcγRIIB-mediated regulation are not known. How FcγRIIB mediates this inhibition to affect B cell homeostasis is also not determined.

Shiang-Jong Tzeng

Graduate Institute of Pharmacology,
College of Medicine,
Graduate Institute of Pharmacology, College of Medicine

National Taiwan University

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Shiang-Jong Tzeng

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National Taiwan University

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Graduate Institute of Pharmacology,
College of Medicine,
Graduate Institute of Pharmacology, College of Medicine