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The CD300 molecules: an   emerging family ofregulators of the Immune         System       Francisco Borrego BioCruces Heal...
Cell surface receptors (NK cells)                          CD300a                                    Vivier et al. 2012. S...
The CD300 gene complexHuman         41920000 41940000                    72500000              72550000                 72...
Human CD300 family of receptorsCD300g           CD300a      CD300b           CD300c            CD300d      CD300e       CD...
The CD300a receptor•   CD300a is broadly expressed across cells of myeloid and lymphoid lineages.    Includes: NK, T cells...
CD300a-Ig binds to cells of low forward scatter                                                                           ...
CD300a-Ig predominantly binds to dead cells                                                                               ...
CD300a-Ig binds to dead cells of different origin                                        Chicken cells                    ...
Specific binding of CD300a-Ig to liposomes                 BIACORE                                    Simhadri et al., 201...
Blocking of CD300a-Ig binding to dead cellsMFG-E8: A soluble protein. Ligand of PS.   Duramycin: A peptide that binds to P...
Modeling of CD300a structure withPhosphatidylserine and Phosphatidylethanolamine                                    Simhad...
Binding of CD300a-Ig mutants to dead cells                                   Simhadri et al., 2012. Blood
CD300a functional recognition of PE           Reporter cell line                                Simhadri et al., 2012. Blood
Human CD300 family of receptorsCD300g           CD300a      CD300b           CD300c            CD300d      CD300e      CD3...
Alignment of Human CD300a with CD300cHu CD300AHu CD300CHu CD300AHu CD300CHu CD300AHu CD300C
Specificity of the antibodies against CD300c    293T Transient Transfection   YTS Stable Cells (Bulk)                     ...
Characterization of CD300c expression in      cells from peripheral blood                              Simhadri et al., 20...
CD300c expression on monocyte-derived cells                               Simhadri et al., 2013. J Innate Immun
Characterization of CD300s’ expression on                        human monocyte-derived dendritic cells and macrophages   ...
Regulation of CD300c expression on monocytes  TLR4 ligand (LPS)          TLR5 ligand (flagellin)                          ...
Binding of CD300c-Ig binding to 7AAD+ve Jurkat           CD300C Binding to 7AAD Jurkat      60                            ...
CD300A vs CD300C            CD300A Binding to 7AAD Jurkat      800                                            LAIR-1 R65K-...
CD300A vs CD300C                        CD300A-Ig binding to pure lipids              1.0              0.8                ...
CD300A vs CD300C       POPS:DOPC (80% PS)   HBS-N+CaCl2          DOPC:POPE (20%PE)                                        ...
CD300f binds to PS                     Choi, Simhadri et al., 2011. J Immunol
LMIR3=CD300F
Functional relevance ofCD300s interaction with lipids
Phagocytosis of apoptotic cells       by macrophages                              Simhadri et al., 2012. Blood
Decreased phagocytosis of apoptotic cells by L929            cells expressing CD300a                                      ...
CD300a down-regulates BCR-mediated activation                  signals         Ca++ Mobilization   NFAT translocation to  ...
CD300a down-regulates BCR-mediated activation                  signals                                     Silva et al., 2...
CD300a inhibits LPS-induced cytokine secretion                from mast cells                                 Nakahashi-Od...
Cross-linking of CD300c delivers activating            signals in monocytes                       TX45                    ...
Co-stimulatory effect of CD300c on LPS treated monocytes                                        Simhadri et al., 2013. J I...
CD300f mediates phagocytosis of apoptotic cells                                 Choi, Simhadri et al., 2011. J Immunol
Human CD300 receptors. Mechanisms of                       Signaling.  CD300g                 CD300a          CD300b      ...
The ITIMs of CD300a are essential for the inhibition of BCR stimulated activation                        Black Line: anti-...
Mechanism of the inhibitory signal     ITAM: Immunoreceptor tyrosine based activating motif.     ITIM: Immunoreceptor tyro...
Proposed model for CD300a mediated         inhibitory signal                                         SHP1                Y...
Tyrosine phosphorylation of CD300a ITIMs     721.221 Cw3: ligand expressing cells.      721.221 Cw6: control cells.       ...
The phosphatases SHP-1 and SHP-2 associate with tyrosine phosphorylated CD300a ITIMs        721.221 Cw3: ligand expressing...
SHP-1, but not SHP-2 or SHIP, is required for CD300a  mediated inhibition of BCR stimulated activation                    ...
SHP-1, but not SHP-2, is required for CD300a                      mediated inhibition of TCR stimulated activation        ...
Model for CD300a mediated inhibitory signal                                       SHP1                                    ...
Clinical relevance of the CD300                 molecules (Mouse)•   CD300a     – Reversal of airway inflammation and remo...
Clinical relevance of the CD300          molecules (Human)•   CD300a/c     –   Novel immunoglobulin superfamily gene clust...
Decreased frequency of CD300a+ B   cells in HIV infected patients                                                         ...
Deregulation of CD300a Expressionon B Cells of HIV Infected Patients                                             HD       ...
Decreased CD300a Expression on Circulating     Mature B Cells From HIV Infected Patients                     Plasma blasts...
The blockade of the CD300a-PS interaction prolongssurvival of mice after Cecal Ligation and Puncture (CLP)                ...
Impaired binding of CD300a-Ig Q94, a SNP linked            to psoriasis susceptibility                                    ...
Potential roles of CD300 molecules  Immature DC              Mature DC                          Gasiorowski et al. 2013. I...
The immuno-modulatory role of CD300a          Stimulating factors:          Antigen recognition                           ...
AcknowledgementsBorrego Lab                   NIAID-RCBS                NIAID-LIR   Venkateswara Simhadri     •   Rodolfo...
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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. 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).
  • Transcript of "The CD300 molecules: an emerging family of regulators of the Immune System"

    1. 1. The CD300 molecules: an emerging family ofregulators of the Immune System Francisco Borrego BioCruces Health Research Institute Ikerbasque Research Professor Basque Foundation for Science
    2. 2. Cell surface receptors (NK cells) CD300a Vivier et al. 2012. Science
    3. 3. The CD300 gene complexHuman 41920000 41940000 72500000 72550000 72600000 72650000 72700000chr17CD300family CD300LG CD300A CD300LB CD300C CD300LD CD300E CD300LFMouse 101900000 101975000 114900000 114950000 115000000 115050000 115100000 chr11 CD300 family CD300lg CD300a LMIR-5 CLM-6 CLM-5 MAIR-II LMIR-7 CLM-2 CD300lf CLM-7 CD300c LMIR-4 LMIR-2 CLM-3 CD300e CD300lb MAIR-IV CLM-4 mIREM-3 Borrego, F. 2013. Blood
    4. 4. Human CD300 family of receptorsCD300g CD300a CD300b CD300c CD300d CD300e CD300f K E E K SHP-1 SHP-2 SHIP PI3K Grb2 DAP12 DAP10Classical ITIM PI3K binding site Mucin-like UnknownNon-classical ITIM Grb-2 binding site FcRγ domain Adaptor Borrego, F. 2013. Blood
    5. 5. The CD300a receptor• CD300a is broadly expressed across cells of myeloid and lymphoid lineages. Includes: NK, T cells, neutrophils, mast cells, and eosinophils, among others. CD300a• The receptor consists of an IgV-like extracellular domain and a cytoplasmic tail that contains three classical and one non-classical ITIMs (S/I/V/LxYxxI/V/L).• Thus far, CD300a has been shown to function exclusively as an inhibitory receptor: – Decreases NK cytotoxic activity from HLA and non-HLA activating receptors (Cantoni et al. 1999. Eur J Immunol). – Suppresses effects of eotaxin, IL-5, and GM-CSF in eosinophils (Munitz et al. 2006. Blood). – Inhibits IgE-mediated degranulation of mast cells (Bachelet et al. 2005. J Immunol.) – We have shown that CD300a inhibits TCR, BCR and FcγRIIa mediated signals, and modulates the phagocytosis of dead cells. • Alvarez et al. 2008. Mol. Immunol. • Narayanan et al. 2010. PLoS One. • Silva et al., 2011. Blood. • Simhadri et al. 2011. BMC Immunol. • Debell et al., 2012. BMC Immunol. • Simhadri et al. 2012. Blood.
    6. 6. CD300a-Ig binds to cells of low forward scatter Low FSC 100 Peripheral Blood Mononuclear Cells 80 (PBMCs) 60 % of Max 40 1000 20 0 0 1 2 3 4 10 10 10 10 10 800 FL1-H High FSC and SSC Scatter High FSC and SSC 100 600 80 SideSSC-H Low FSC 60 % of Max 400 40 % of Maximum 20 200 0 100 101 102 103 104 Medium FSC Medium FSC FL1-H 0 100 0 200 400 600 800 1000 FSC-H Forward Scatter 80 60 % of Max 40 20 0 100 101 102 103 104 Ig-AF488 FL1-H: Ig-488 Simhadri et al., 2012. Blood
    7. 7. CD300a-Ig predominantly binds to dead cells Late Apoptotic 100 80 60 % of Max 104 40 Late Apoptotic 20 3 10 0 100 Early Apoptotic 101 102 FL1-H: Ig-488 103 104 FL3-H: 7-AAD 100 Live Early Apoptotic 7-AAD 2 80 10 60 % of Max 40 1 10 % of Maximum 20 0 100 101 102 103 104 10 0 Live FL1-H: Ig-488 100 101 102 103 104 100 Annexin V FL4-H: Annexin V 80 60 % of Max 40 20 0 0 1 2 3 4 10 10 10 10 10 FL1-H: Ig-488 Ig-AF488 Simhadri et al., 2012. Blood
    8. 8. CD300a-Ig binds to dead cells of different origin Chicken cells Insect cells Simhadri et al., 2012. Blood
    9. 9. Specific binding of CD300a-Ig to liposomes BIACORE Simhadri et al., 2012. Blood
    10. 10. Blocking of CD300a-Ig binding to dead cellsMFG-E8: A soluble protein. Ligand of PS. Duramycin: A peptide that binds to PE. Simhadri et al., 2012. Blood
    11. 11. Modeling of CD300a structure withPhosphatidylserine and Phosphatidylethanolamine Simhadri et al., 2012. Blood
    12. 12. Binding of CD300a-Ig mutants to dead cells Simhadri et al., 2012. Blood
    13. 13. CD300a functional recognition of PE Reporter cell line Simhadri et al., 2012. Blood
    14. 14. Human CD300 family of receptorsCD300g CD300a CD300b CD300c CD300d CD300e CD300f K E E K SHP-1 SHP-2 SHIP PI3K Grb2 DAP12 DAP10Classical ITIM PI3K binding site Mucin-like UnknownNon-classical ITIM Grb-2 binding site FcRγ domain Adaptor Borrego, F. 2013. Blood
    15. 15. Alignment of Human CD300a with CD300cHu CD300AHu CD300CHu CD300AHu CD300CHu CD300AHu CD300C
    16. 16. Specificity of the antibodies against CD300c 293T Transient Transfection YTS Stable Cells (Bulk) Simhadri et al., 2013. J Innate Immun
    17. 17. Characterization of CD300c expression in cells from peripheral blood Simhadri et al., 2013. J Innate Immun
    18. 18. CD300c expression on monocyte-derived cells Simhadri et al., 2013. J Innate Immun
    19. 19. Characterization of CD300s’ expression on human monocyte-derived dendritic cells and macrophages CD300a/c CD300c CD300LE CD300LFPercentage of Maximum Immature Dendritic cells Mature Dendritic cells Macrophages Expression
    20. 20. Regulation of CD300c expression on monocytes TLR4 ligand (LPS) TLR5 ligand (flagellin) Simhadri et al., 2013. J Innate Immun
    21. 21. Binding of CD300c-Ig binding to 7AAD+ve Jurkat CD300C Binding to 7AAD Jurkat 60 LAIR-1 R65K-Ig CD300C-Ig 40MFI 20 0 0 20 40 60 Ig-protein (µg/ml) Simhadri et al., (manuscript in preparation)
    22. 22. CD300A vs CD300C CD300A Binding to 7AAD Jurkat 800 LAIR-1 R65K-Ig CD300A-Ig 600MFI 400 200 0 0 20 40 60 Ig-protein (µg/ml) CD300C Binding to 7AAD Jurkat 60 LAIR-1 R65K-Ig CD300C-Ig 40MFI 20 0 0 20 40 60 Ig-protein (µg/ml) Simhadri et al., (manuscript in preparation)
    23. 23. CD300A vs CD300C CD300A-Ig binding to pure lipids 1.0 0.8 CD300A-PSOD (450 nm) CD300A-PE 0.6 CD300A-PC LAIR1 R65K-PS 0.4 LAIR1 R65K-PE LAIR1 R65K-PC 0.2 0.0 0 20 40 60 80 Ig-protein (µg/ml) CD300C-Ig binding to pure lipids 0.6 CD300C-PS CD300C-PEOD (450 nm) CD300C-PC 0.4 LAIR1 R65K-PS LAIR1 R65K-PE LAIR1 R65K-PC 0.2 0.0 0 20 40 60 80 Ig-protein (µg/ml) Simhadri et al., (manuscript in preparation)
    24. 24. CD300A vs CD300C POPS:DOPC (80% PS) HBS-N+CaCl2 DOPC:POPE (20%PE) HBS-N+CaCl2400 LAIR1 R65K 2000 LAIR1 R65K CD300A300 CD300C 1500 CD300A CD300C200 1000100 500 0 0-100 -500 POPS:DOPC DOPC: POPE Simhadri et al., (manuscript in preparation)
    25. 25. CD300f binds to PS Choi, Simhadri et al., 2011. J Immunol
    26. 26. LMIR3=CD300F
    27. 27. Functional relevance ofCD300s interaction with lipids
    28. 28. Phagocytosis of apoptotic cells by macrophages Simhadri et al., 2012. Blood
    29. 29. Decreased phagocytosis of apoptotic cells by L929 cells expressing CD300a Simhadri et al., 2012. Blood
    30. 30. CD300a down-regulates BCR-mediated activation signals Ca++ Mobilization NFAT translocation to the nucleus Silva et al., 2011. Blood
    31. 31. CD300a down-regulates BCR-mediated activation signals Silva et al., 2011. Blood
    32. 32. CD300a inhibits LPS-induced cytokine secretion from mast cells Nakahashi-Oda et al. 2012. J Exp Med
    33. 33. Cross-linking of CD300c delivers activating signals in monocytes TX45 MOPC-21 Unstimulated Simhadri et al., 2013. J Innate Immun
    34. 34. Co-stimulatory effect of CD300c on LPS treated monocytes Simhadri et al., 2013. J Innate Immun
    35. 35. CD300f mediates phagocytosis of apoptotic cells Choi, Simhadri et al., 2011. J Immunol
    36. 36. Human CD300 receptors. Mechanisms of Signaling. CD300g CD300a CD300b CD300c CD300d CD300e CD300f K E E K SHP-1 SHP-2 SHIP PI3K Grb2 DAP12 DAP10Classical ITIM PI3K binding site Mucin-like UnknownNon-classical ITIM Grb-2 binding site FcRγ domain Adaptor Borrego, F. 2013. Blood
    37. 37. The ITIMs of CD300a are essential for the inhibition of BCR stimulated activation Black Line: anti-BCRCa++ Mobilization Grey Line: anti-BCR + anti-CD300a WT 4F Y F Y FNFAT translocation to Y F the nucleus Y F Debell et al., 2012. BMC Immunol
    38. 38. Mechanism of the inhibitory signal ITAM: Immunoreceptor tyrosine based activating motif. ITIM: Immunoreceptor tyrosine based inhibitory motif. Long, E.O. 2008. Annu Rev Immunol
    39. 39. Proposed model for CD300a mediated inhibitory signal SHP1 Y Y SHP2 Y Y SHIP YP YP YP YP YP YP YP YP YP YP YP YP SHP1 SHP2 SHIP Inhibitory signal
    40. 40. Tyrosine phosphorylation of CD300a ITIMs 721.221 Cw3: ligand expressing cells. 721.221 Cw6: control cells. Debell et al., 2012. BMC Immunol
    41. 41. The phosphatases SHP-1 and SHP-2 associate with tyrosine phosphorylated CD300a ITIMs 721.221 Cw3: ligand expressing cells. 721.221 Cw6: control cells. Debell et al., 2012. BMC Immunol
    42. 42. SHP-1, but not SHP-2 or SHIP, is required for CD300a mediated inhibition of BCR stimulated activation Black Line: anti-BCR Grey Line: anti-BCR + anti-CD300a *** NFAT translocation to the nucleus Debell et al., 2012. BMC Immunol
    43. 43. SHP-1, but not SHP-2, is required for CD300a mediated inhibition of TCR stimulated activation 2.0 SHP-1 mRNA SHP-2 mRNA 1.5 1.0 0.5t nuo mA evt a e R 0.0 A A A A A A N N N N N N i l SH siR SH siR R R R R si si si si T T 1 2 1 2 P- P- P- P- N N SH SH Debell et al., 2012. BMC Immunol
    44. 44. Model for CD300a mediated inhibitory signal SHP1 ??? Y Y SHP2 Y ??? Y SHIP YP YP YP YP YP YP YP YP YP YP YP YP YP YP YP YP SHP1 SHP2 SHIP ??? Inhibitory signal ?
    45. 45. Clinical relevance of the CD300 molecules (Mouse)• CD300a – Reversal of airway inflammation and remodeling in asthma by a bispecific antibody fragment linking CCR3 to CD300a (Munitz et al., J Allergy Clin. Immunol., 2006). – Abrogation of allergic reactions by a bispecific antibody fragment linking IgE to CD300a (Bachelet et al., J Allergy Clin. Immunol., 2006). – Suppression of Normal and Malignant Kit Signaling by a Bispecific Antibody Linking Kit with CD300a (Bachelet et al., J. Immunol., 2008). – Apoptotic cells suppress mast cell inflammatory responses via the CD300a immunoreceptor (Nakahashi-Oda et al., J. Exp. Med., 2012).• CD300b – CD300b deficiency ameliorates mouse kidney ischemia/reperfusion injury (Yamanishi et al., J. Exp. Med., 2010). – A soluble form of LMIR5/CD300b amplifies lipopolysaccharide-induced lethal inflammation in sepsis (Yamanishi et al., J. Immunol. 2012).• CD300lf – Negative regulation of autoimmune demyelination by the inhibitory receptor CD300lf (Xi et al., J. Exp. Med., 2009). – Overexpression of the immunoreceptor CD300f has a neuroprotective role in a model of acute brain injury (Peluffo et al., Brain Pathol, 2011). – The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding extracellular ceramide (Izawa et al., Immunity, 2012).
    46. 46. Clinical relevance of the CD300 molecules (Human)• CD300a/c – Novel immunoglobulin superfamily gene cluster, mapping to a region of human chromosome 17q25, linked to psoriasis susceptibility (Speckman et al., Hum. Genet., 2003). – Genetic evidence implicates the immune system and cholesterol metabolism in the aetiology of Alzheimers disease (Jones et al., PLoS One, 2010). – Human Th1 cells that express CD300a are polyfunctional and after stimulation up-regulate the T-box transcription factor eomesodermin (Narayanan et al., PLoS One, 2010). – Differential Expression of CD300a/c on Human TH1 and TH17 cells (Simhadri et al., BMC Immunol, 2011). – Blood-based Biomarkers Can Differentiate Ulcerative Colitis from Crohn’s Disease and Noninflammatory Diarrhea (Burakoff et al., Inflamm. Bowel Dis., 2011). – New markers for minimal residual disease detection in acute lymphoblastic leukemia (Coustan- Smith et al., Blood, 2011). – CD300a is expressed on human B cells, modulates BCR mediated signaling and its expression is down-regulated in HIV infection (Silva et al., Blood, 2011).
    47. 47. Decreased frequency of CD300a+ B cells in HIV infected patients 50 ** % of CD300a+ B cells 40 ** ns 30 20 10 0 HD HIV-AVIR HIV-VIR ** 60 *** ns 50 * in the CD21+ subset % of CD300a+ cellsin the CD21- subset% of CD300a+ cells 40 ** ns 40 30 20 20 10 0 0 HD HIV-AVIR HIV-VIR HD HIV-AVIR HIV-VIR Silva et al., 2011. Blood
    48. 48. Deregulation of CD300a Expressionon B Cells of HIV Infected Patients HD HIV-AVIR HIV-VIR 5 4.9 25 5 1.9 6.6 5 6.3 3.6 10 10 10 <PE-A>: CD300a <PE-A>: CD300a <PE-A>: CD300a 4 4 4CD300a 10 10 10 3 3 3 10 10 10 0 0 0 4 66 22 69 48 42 3 4 5 3 4 5 3 4 5 0 10 10 10 0 10 10 10 0 10 10 10 <FITC-A>: CD21 <FITC-A>: CD21 <FITC-A>: CD21 CD21 Silva et al., 2011. Blood
    49. 49. Decreased CD300a Expression on Circulating Mature B Cells From HIV Infected Patients Plasma blasts Resting Memory Activated Memory 8000 ** 1500 ** 800 *** ns *** *** * nsCD300a MFI 6000 600 ns 1000 4000 400 500 2000 200 0 0 0 HD HIV-AVIR HIV-VIR HD HIV-AVIR HIV-VIR HD HIV-AVIR HIV-VIR Naive Atypical Memory (exhausted) ns ** * 500 ns *** 400 ns 400 CD300a MFI 300 300 200 200 100 100 0 0 HD HIV-AVIR HIV-VIR HD HIV-AVIR HIV-VIR Silva et al., 2011. Blood
    50. 50. The blockade of the CD300a-PS interaction prolongssurvival of mice after Cecal Ligation and Puncture (CLP) Nakahashi-Oda et al. 2012. J Exp Med
    51. 51. Impaired binding of CD300a-Ig Q94, a SNP linked to psoriasis susceptibility Simhadri et al., 2012. Blood
    52. 52. Potential roles of CD300 molecules Immature DC Mature DC Gasiorowski et al. 2013. Immunol Letters
    53. 53. The immuno-modulatory role of CD300a Stimulating factors: Antigen recognition CD300a n Modulatory function by io Danger signals t nc creating anti-inflammatory Fu Inflammatory milieu environment ry Oncogenic Transformation to bi hi InNaïve/resting ActivatedImmune Immune SystemSystem Shut Down Removal of Activated Cells: Apoptosis and subsequent Eradication of Insult phagocytosis (Macrophages). Cytotoxicity Cytolysis (NK cells). Pro-inflammatory cytokines Activated cells express ligands for receptors expressed on NK cells and macrophages.
    54. 54. AcknowledgementsBorrego Lab NIAID-RCBS NIAID-LIR Venkateswara Simhadri • Rodolfo Silva  Susan Moir Karen Debell • Seung Choi • Linjie Tian  Lela Kardava John Mariano • John E. Coligan Qing Zhou Aleksandra Gil-Krzewska Milena Dimitrova University of Sevilla University of Córdoba Manuel Leal  José Peña Sara Ferrando-Martínez

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