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The CD300 molecules: an emerging family of regulators of the Immune System
 

The CD300 molecules: an emerging family of regulators of the Immune System

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In order to provide an adequate response that allows the elimination of insults while preserving self, the immune system is tightly regulated by a balance between activating and inhibitory signals. ...

In order to provide an adequate response that allows the elimination of insults while preserving self, the immune system is tightly regulated by a balance between activating and inhibitory signals. Multiple mechanisms exist to accomplish this task, including the expression of activating and inhibitory receptors by immune cells. The CD300 family of receptors are type I transmembrane proteins that forms an arrayed receptor system that is able to recognize the viability and activation status of cells, and consequently have a significant influence on the final outcome of the immune response. The very recent discovery that CD300 molecules are able to recognize lipids, such as phosphatidylserine, and phosphatidylethanolamine that are exposed on the outer leaflet of the plasma membrane of dead and activated cells has opened a new field of research. Through their binding to lipids and other ligands, this family of receptors is poised to have a significant role in complex biological processes and in the host response to severe pathological conditions. Expression of CD300 molecules is altered in a number of diseases and anti-CD300 antibodies have been demonstrated to have significant therapeutic effect in several animal models. The mechanisms underlying the immunoregulatory effects of the CD300 family are complex and deciphering their signaling properties will allow effective targeting of these molecules as novel therapies in a wide variety of inflammatory and immune-mediated diseases.

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  • La gran parte de mi vida como investigador la he dedicado al estudio de las celulas NK y especialmente a los receptores de superficie. Como ven en esta diapositiva, las celulas NK, como cualquier otra celula, estan equipados con una bateria de receptores activadores e inhibidores, ademas de receptores para chemokinas, cytokinas y de adhesion. En el laboratorio del Dr. Coligan, descubrimos los receptores CD94/NKG2, su ligando HLA-E. Tambien hemos trabajado en otros receptores como los KIRs y NKG2D. Desde que me mude a la FDA y empeze con mi propio laboratorio, mi grupo esta dedicado a otro grupo de receptores, la familia CD300, y fundamentalmente al estudio de CD300a, un receptor inhibidor, que inicialmente se describio en las celulas NK.
  • All have an extracellular IgV-like domain with 2 disulfide bonds. The search for ligands for the CD300 family members is an active area of research. They are expressed on cell of both lymphoid and myeloid lineages. There are activating (with short intracellular tail) and inhibitory (with long intracellular tail) members.
  • As I said earlier that the expression of CD300a is confined to both lymphoid and myeloid lineages, we predicted that the function of this receptor should be more localized. So we have decided to use PBLs as our source to fish out the ligand. For this we have used a recombinant fusion protein CD300a-Ig. The flow cytometry data depicts that the fusion protein bound to a cell population that is in the low forward and side scatter dot plot. The red histograms display the binding of LAIR1-Ig i.e. used as a negative control all through my experiments. Presuming that the low FSC to dead population we have next analyzed in detail the viability of the cells and checked for the binding.
  • When the cells were stained for Annexin V and 7-AAD to differentiate between the early and late apoptotic (necrotic) cells, we found that the CD300a-Ig predominantly bound to the double positive population i.e. the late apoptotic cells.
  • When the cells were stained for Annexin V and 7-AAD to differentiate between the early and late apoptotic (necrotic) cells, we found that the CD300a-Ig predominantly bound to the double positive population i.e. the late apoptotic cells.
  • We have generated a reporter cell line with the extracellular domain of the receptor and the cytoplasmic tail of CD3 zeta. Upon ligation to the receptor, the CD3 zeta gets phosphorylated and signals the synthesis of beta-galacosidase. And thus the beta-gal activity is measured.
  • CD300a inhibits LPS-induced cytokine secretion from mast cells. (B–D) WT or Cd300a −/− BMMCs mixed with apoptotic cells at a ratio of 1:0.1 were stimulated with 1 µg/ml LPS for 4 h. The culture supernatant was subjected to proteome analyses (B) or ELISA (C; n = 6) in the absence (B and C) or presence (C) of D89E MFG-E8. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01, Student's t test. Error bars show SD.
  • (A) DT40 chicken B cells expressing CD300a WT or CD300a 4F were loaded with Fluo-4 and Fura-Red. Cells were stimulated with anti-chicken BCR plus isotype control antibody (black line) or anti-chicken BCR plus anti-CD300a mAb (grey line) for 30 seconds and then co-crosslinked with a secondary antibody (GAM). Fluorescence emission was measured in a flow cytometer. Ca2+ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. These results are representative of three independent experiments. (B) DT40 chicken B cells expressing CD300a WT or CD300a 4F were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with GAM plus anti-chicken BCR plus isotype control or anti-chicken BCR plus anti-CD300a mAb. The measured luciferase activity was normalized to the activity obtained with cells treated with PMA plus ionomycin. Data are presented as percentage of inhibition of CD300a vs. isotype control and they are the average ± SEM for three separate experiments.
  • Binding of inhibitory receptor to its ligand on a target cell is sufficient to induce receptor clustering. Two tyrosines, each within a cytoplasmic ITIM sequence, are phosphorylated by an Src family kinase. Tyrosine-phosphorylated ITIMs recruit and activate the tyrosine phosphatase SHP-1. Catalytically active SHP-1 dephosphorylates multiple substrates, such as activation receptors and signaling molecules, to prevent NK cell cytotoxicity.
  • Varias autores han publicado que CD300a se una a las fosfatasas SHP-1, SHP-2 y SHIP cuando se fosforila y especulan que todas estas fosfatasas son las responsables de la senyal negativa mediada a traves de CD300a. Sin embargo, es evidente que union no significa directamente senyal negativa y eso hay que demostrarlo. Karen decidio hacerlo.
  • KIR-CD300a WT and KIR-CD300a 4F Jurkat T cells were stimulated with medium or pervanadate for 3 minutes, or mixed with 721.221-Cw3 or 721.221-Cw6 and incubated at 37oC for 5 minutes. Cell lysates were immunopreciprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for phosphotyrosine and HA. Results are representative of five independent experiments.
  • KIR-CD300a Jurkat T cells were stimulated with medium or pervanadate for 3 minutes, or incubated for 5 minutes at 37oC with 721.221-Cw3 and 721.221-Cw6 cells. Then, cell lysates were immunopreciprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for HA, SHP-1 and SHP-2. Results are representative of two independent experiments.
  • (A) DT40 cells, DT40 cells lacking SHP-1, DT40 cells lacking SHP-2, or DT40 cells lacking SHIP, all expressing CD300a WT were loaded with Fluo-4 and Fura-Red. Then, cells were acquired in a flow cytometer and stimulated with anti-chicken BCR plus isotype control antibody (black line) or anti-chicken BCR plus anti-CD300a (grey line) mAb for 30 seconds and then co-crosslinked with a secondary antibody (GAM). Ca2+ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. These results are representative of two independent experiments. (B) DT40 cell lines expressing CD300a WT were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with anti-chicken BCR plus isotype control or anti-chicken BCR plus anti-CD300a mAb. Cells were lysed and supernatants assayed for luciferase activity. Results were normalized to the activity obtained when cell were treated with PMA plus ionomycin. Data are presented as percentage of inhibition of CD300a vs. isotype control and they are the average ± SEM for three separate experiments.
  • Este es el modelo con el que nosotros estamos trabajando ahora. Describe el modelo sin olvidar mencionar que SHP-2 es posible que tenga un papel activador.
  • La relevancia clinica de los receptores CD300 se esta descubriendo muy recientemente.
  • La relevancia clinica de los receptores CD300 se esta descubriendo muy recientemente.
  • PBMCs from healthy donors (HD), HIV-aviremic (HIV-AVIR) and HIV-viremic (HIV-VIR) patients were labeled with anti-CD10, anti-CD19, anti-CD20, anti-CD21, anti-CD27 and anti-CD300a mAb. (Left) , the percentage of CD300a+ cells B cells is shown. Each symbol represents a different donor. (Below) , the percentage of CD300a+ cells among CD21+ B cells (left panel) and CD21- B cells (right panel) was determined. Samples were acquired in FACS Canto from BD Biosciences, and analyzed with the FlowJo software.
  • PBMCs from healthy donors (HD), HIV aviremic (HIV-AVIR) and HIV viremic (HIV-VIR) patients were labeled with anti-CD10, anti-CD19, anti-CD20, anti-CD21, anti-CD27 and anti-CD300a mAb. The lymphocyte gate was determined according to the forward and side scatter parameters. Representative dot plots of anti-CD21 and CD300a mAb staining in the CD19+ gate from HD, HIV-AVIR and HIV-VIR.
  • The blockade of the CD300a–PS interaction prolongs survival of mice after CLP. (A–C) WT mice were injected i.p. with 500 µg of control antibody ( n = 11) or anti-CD300a monoclonal antibody (TX41; n = 13), 1 h before and 18 h after CLP, and the survival rate is shown (A). Bacterial CFUs (B) and the numbers of neutrophils (C) in the peritoneal lavage fluid of mice ( n = 5 in each group) were determined 4 h after CLP, as described. (D and E) WT or Cd300 a −/− mice were injected i.p. with 50 µg D89E MFG-E8 ( n = 10 and 8, respectively) or PBS ( n = 9), 1 h before and 18 h after CLP, and the survival rate is shown (D). Bacterial CFUs in the peritoneal lavage fluid of mice ( n = 4 in each group) were determined 4 h after CLP, as described (E). *, P < 0.05, Student's t test. Error bars show SD. Data in A and D were each pooled from two independent experiments.
  • Fig. 2. Schematic of the potential roles CD300 molecules can play in DC biology. (A) In immature DC, CD300 molecules can inhibit TLR signalling (1) which downregulates CD300 molecules via a feedback loop (2). CD300 molecules have been shown to both inhibit and stimulate phagocytosis (3 and 4). CD300 molecules can upregulate chemokine receptors resulting in enhanced migration (5). CD300 molecules are expressed on NK cells resulting in a further potential mechanism by which they may influence DC (6). (B) In mature DC inhibitory CD300 molecules downregulate antigen specific T cell responses (1). They downregulate HLA-DR (2) whilst other cytokines such as IL-6 are increased (3). TNF and IFN-α are decreased (4) and IFN-α can downregulate CD300 a/c via a feedback loop (5). Triggering CD300 molecules upregulate co-stimulatory molecules including CD40 (6) and can increase TNF (7).

The CD300 molecules: an emerging family of regulators of the Immune System The CD300 molecules: an emerging family of regulators of the Immune System Presentation Transcript