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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Emerging regulators of the immune system
1. The CD300 molecules: an
emerging family of
regulators of the Immune
System
Francisco Borrego
BioCruces Health Research Institute
Ikerbasque Research Professor
Basque Foundation for Science
4. Human CD300 family of receptors
CD300g CD300a CD300b CD300c CD300d CD300e CD300f
K E E K
SHP-1 SHP-2 SHIP PI3K Grb2 DAP12 DAP10
Classical ITIM PI3K binding site
Mucin-like Unknown
Non-classical ITIM Grb-2 binding site FcRγ
domain Adaptor
Borrego, F. 2013. Blood
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. 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. 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. CD300a-Ig binds to dead cells of different origin
Chicken cells
Insect cells
Simhadri et al., 2012. Blood
9. Specific binding of CD300a-Ig to liposomes
BIACORE
Simhadri et al., 2012. Blood
10. Blocking of CD300a-Ig binding to dead cells
MFG-E8: A soluble protein. Ligand of PS. Duramycin: A peptide that binds to PE.
Simhadri et al., 2012. Blood
11. Modeling of CD300a structure with
Phosphatidylserine and Phosphatidylethanolamine
Simhadri et al., 2012. Blood
14. Human CD300 family of receptors
CD300g CD300a CD300b CD300c CD300d CD300e CD300f
K E E K
SHP-1 SHP-2 SHIP PI3K Grb2 DAP12 DAP10
Classical ITIM PI3K binding site
Mucin-like Unknown
Non-classical ITIM Grb-2 binding site FcRγ
domain Adaptor
Borrego, F. 2013. Blood
15. Alignment of Human CD300a with CD300c
Hu CD300A
Hu CD300C
Hu CD300A
Hu CD300C
Hu CD300A
Hu CD300C
16. Specificity of the antibodies against CD300c
293T Transient Transfection YTS Stable Cells (Bulk)
Simhadri et al., 2013. J Innate Immun
17. Characterization of CD300c expression in
cells from peripheral blood
Simhadri et al., 2013. J Innate Immun
18. CD300c expression on monocyte-derived cells
Simhadri et al., 2013. J Innate Immun
19. Characterization of CD300s’ expression on
human monocyte-derived dendritic cells and macrophages
CD300a/c CD300c CD300LE CD300LF
Percentage of Maximum
Immature
Dendritic cells
Mature
Dendritic cells
Macrophages
Expression
20. Regulation of CD300c expression on monocytes
TLR4 ligand (LPS) TLR5 ligand (flagellin)
Simhadri et al., 2013. J Innate Immun
21. Binding of CD300c-Ig binding to 7AAD+ve Jurkat
CD300C Binding to 7AAD Jurkat
60
LAIR-1 R65K-Ig
CD300C-Ig
40
MFI
20
0
0 20 40 60
Ig-protein (µg/ml)
Simhadri et al., (manuscript in preparation)
22. CD300A vs CD300C
CD300A Binding to 7AAD Jurkat
800
LAIR-1 R65K-Ig
CD300A-Ig
600
MFI
400
200
0
0 20 40 60
Ig-protein (µg/ml)
CD300C Binding to 7AAD Jurkat
60
LAIR-1 R65K-Ig
CD300C-Ig
40
MFI
20
0
0 20 40 60
Ig-protein (µg/ml)
Simhadri et al., (manuscript in preparation)
23. CD300A vs CD300C
CD300A-Ig binding to pure lipids
1.0
0.8 CD300A-PS
OD (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-PE
OD (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. CD300A vs CD300C
POPS:DOPC (80% PS) HBS-N+CaCl2 DOPC:POPE (20%PE)
HBS-N+CaCl2
400 LAIR1 R65K 2000
LAIR1 R65K
CD300A
300 CD300C 1500 CD300A
CD300C
200 1000
100 500
0 0
-100 -500
POPS:DOPC DOPC: POPE
Simhadri et al., (manuscript in preparation)
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 DAP10
Classical ITIM PI3K binding site
Mucin-like Unknown
Non-classical ITIM Grb-2 binding site FcRγ
domain Adaptor
Borrego, F. 2013. Blood
37. The ITIMs of CD300a are essential for the
inhibition of BCR stimulated activation
Black Line: anti-BCR
Ca++ Mobilization Grey Line: anti-BCR + anti-CD300a
WT 4F
Y F
Y F
NFAT translocation to Y F
the nucleus Y F
Debell et al., 2012. BMC Immunol
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. 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. Tyrosine phosphorylation of CD300a ITIMs
721.221 Cw3: ligand expressing cells.
721.221 Cw6: control cells.
Debell et al., 2012. BMC Immunol
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. 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. 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
0.5
t 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. 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. 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. 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
Alzheimer's 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. 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+ cells
in 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
49. Decreased CD300a Expression on Circulating
Mature B Cells From HIV Infected Patients
Plasma blasts Resting Memory Activated Memory
8000 ** 1500 ** 800 ***
ns *** ***
* ns
CD300a 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. The blockade of the CD300a-PS interaction prolongs
survival of mice after Cecal Ligation and Puncture (CLP)
Nakahashi-Oda et al. 2012. J Exp Med
51. Impaired binding of CD300a-Ig Q94, a SNP linked
to psoriasis susceptibility
Simhadri et al., 2012. Blood
52. Potential roles of CD300 molecules
Immature DC Mature DC
Gasiorowski et al. 2013. Immunol Letters
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
In
Naïve/resting Activated
Immune Immune System
System 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. Acknowledgements
Borrego 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
Notas do Editor
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).