the cd300 molecules: an emerging family of regulators of the immune system
DESCRIPTION
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.TRANSCRIPT
The CD300 molecules: an emerging family of
regulators of the Immune System
Francisco BorregoBioCruces Health Research Institute
Ikerbasque Research ProfessorBasque Foundation for Science
Cell surface receptors (NK cells)
CD300a
Vivier et al. 2012. Science
chr17
CD300
family
72500000 72550000 72600000 72650000 72700000
CD300A CD300LB CD300C CD300E CD300LFCD300LD
41920000 41940000
CD300LG
Human
101975000101900000
CD300lg
chr11
CD300
family
114900000 114950000 115000000 115050000 115100000
CD300a LMIR-5
CLM-7
CD300lb
mIREM-3
CLM-6
CD300c
CLM-2
CD300e
CD300lfMAIR-II
LMIR-2
CLM-4
LMIR-7
CLM-3
Mouse
CLM-5
LMIR-4
MAIR-IV
Borrego, F. 2013. Blood
The CD300 gene complex
Classical ITIM
Non-classical ITIM
K KEE
CD300a CD300b CD300c CD300d CD300e CD300f
PI3K binding site
Grb-2 binding site
DAP12
FcRγ
DAP10
Mucin-likedomain
UnknownAdaptor
SHP-1 SHP-2 SHIP
CD300g
PI3K Grb2
Borrego, F. 2013. Blood
Human CD300 family of receptors
• CD300a is broadly expressed across cells of myeloid and lymphoid lineages. Includes: NK, T cells, neutrophils, mast cells, and eosinophils, among others.
• 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.
CD300a
The CD300a receptor
Forward Scatter
Sid
e S
catt
er
Low FSC
High FSC and SSC
Medium FSC
Low FSC
High FSC and SSC
Ig-AF488
% o
f M
axim
um
Medium FSC
CD300a-Ig binds to cells of low forward scatter
Peripheral Blood Mononuclear Cells (PBMCs)
Simhadri et al., 2012. Blood
Annexin V
7-A
AD Live
Late Apoptotic
Early Apoptotic
Late Apoptotic
Early Apoptotic
Ig-AF488
% o
f M
axim
um
Live
CD300a-Ig predominantly binds to dead cells
Simhadri et al., 2012. Blood
CD300a-Ig binds to dead cells of different origin
Chicken cells
Insect cells
Simhadri et al., 2012. Blood
Specific binding of CD300a-Ig to liposomes BIACORE
Simhadri et al., 2012. Blood
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
Modeling of CD300a structure with Phosphatidylserine and Phosphatidylethanolamine
Simhadri et al., 2012. Blood
Binding of CD300a-Ig mutants to dead cells
Simhadri et al., 2012. Blood
CD300a functional recognition of PEReporter cell line
Simhadri et al., 2012. Blood
Classical ITIM
Non-classical ITIM
K KEE
CD300a CD300b CD300c CD300d CD300e CD300f
PI3K binding site
Grb-2 binding site
DAP12
FcRγ
DAP10
Mucin-likedomain
UnknownAdaptor
SHP-1 SHP-2 SHIP
CD300g
PI3K Grb2
Human CD300 family of receptors
Borrego, F. 2013. Blood
Hu CD300AHu CD300C
Hu CD300AHu CD300C
Hu CD300AHu CD300C
Alignment of Human CD300a with CD300c
293T Transient Transfection YTS Stable Cells (Bulk)
Specificity of the antibodies against CD300c
Simhadri et al., 2013. J Innate Immun
Characterization of CD300c expression in cells from peripheral blood
Simhadri et al., 2013. J Innate Immun
CD300c expression on monocyte-derived cells
Simhadri et al., 2013. J Innate Immun
Per
cent
age
of M
axim
um
CD300a/c CD300c CD300LE CD300LF
Characterization of CD300s’ expression on human monocyte-derived dendritic cells and macrophages
Immature Dendritic cells
Mature Dendritic cells
Macrophages
Expression
Regulation of CD300c expression on monocytes
TLR4 ligand (LPS) TLR5 ligand (flagellin)
Simhadri et al., 2013. J Innate Immun
Binding of CD300c-Ig binding to 7AAD+ve Jurkat
Simhadri et al., (manuscript in preparation)
CD300A vs CD300C
Simhadri et al., (manuscript in preparation)
CD300A vs CD300C
Simhadri et al., (manuscript in preparation)
CD300A vs CD300C
Simhadri et al., (manuscript in preparation)
CD300f binds to PS
Choi, Simhadri et al., 2011. J Immunol
LMIR3=CD300F
Functional relevance of CD300s interaction with lipids
Phagocytosis of apoptotic cells by macrophages
Simhadri et al., 2012. Blood
Decreased phagocytosis of apoptotic cells by L929 cells expressing CD300a
Simhadri et al., 2012. Blood
CD300a down-regulates BCR-mediated activation signals
Ca++ Mobilization NFAT translocation to the nucleus
Silva et al., 2011. Blood
CD300a down-regulates BCR-mediated activation signals
Silva et al., 2011. Blood
Nakahashi-Oda et al. 2012. J Exp Med
CD300a inhibits LPS-induced cytokine secretion from mast cells
Cross-linking of CD300c delivers activating signals in monocytes
TX45
MOPC-21
Unstimulated
Simhadri et al., 2013. J Innate Immun
Co-stimulatory effect of CD300c on LPS treated monocytes
Simhadri et al., 2013. J Innate Immun
CD300f mediates phagocytosis of apoptotic cells
Choi, Simhadri et al., 2011. J Immunol
Classical ITIM
Non-classical ITIM
K KEE
CD300a CD300b CD300c CD300d CD300e CD300f
PI3K binding site
Grb-2 binding site
DAP12
FcRγ
DAP10
Mucin-likedomain
UnknownAdaptor
SHP-1 SHP-2 SHIP
CD300g
PI3K Grb2
Human CD300 receptors. Mechanisms of Signaling.
Borrego, F. 2013. Blood
The ITIMs of CD300a are essential for the inhibition of BCR stimulated activation
Ca++ Mobilization
NFAT translocation to the nucleus
YYY
Y
FFF
F
WT 4F
Debell et al., 2012. BMC Immunol
Black Line: anti-BCRGrey Line: anti-BCR + anti-CD300a
Mechanism of the inhibitory signal
ITAM: Immunoreceptor tyrosine based activating motif.
ITIM: Immunoreceptor tyrosine based inhibitory motif.Long, E.O. 2008. Annu Rev Immunol
SHP1
YYYY
SHP1
P
PP
P
YYYY
SHP2
YYYY
SHP2
P
PP
P
SHIP
YYYY
SHIP
P
PP
P
Proposed model for CD300a mediated inhibitory signal
Inhibitory signal
Tyrosine phosphorylation of CD300a ITIMs
Debell et al., 2012. BMC Immunol
721.221 Cw3: ligand expressing cells.
721.221 Cw6: control cells.
The phosphatases SHP-1 and SHP-2 associate with tyrosine phosphorylated CD300a ITIMs
Debell et al., 2012. BMC Immunol
721.221 Cw3: ligand expressing cells.
721.221 Cw6: control cells.
SHP-1, but not SHP-2 or SHIP, is required for CD300a mediated inhibition of BCR stimulated activation
Debell et al., 2012. BMC Immunol
NFAT translocation to the nucleus
Black Line: anti-BCRGrey Line: anti-BCR + anti-CD300a
***
SHP-1, but not SHP-2, is required for CD300a mediated inhibition of TCR stimulated activation
NT siR
NA
SHP-1 s
iRNA
SHP-2 s
iRNA
NT siR
NA
SHP-1 s
iRNA
SHP-2 s
iRNA
0.0
0.5
1.0
1.5
2.0 SHP-1 mRNASHP-2 mRNA
Rel
ativ
e A
mou
nt
Debell et al., 2012. BMC Immunol
SHP1
YYYY
SHP1
P
PP
P
YYYY
SHP2
YYYY
SHP2
P
PP
P
SHIP
YYYY
SHIP
P
PP
P
Model for CD300a mediated inhibitory signal
???
???
Inhibitory signal ?
YYYY
???
PPP
P
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).
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).
HD HIV-AVIR HIV-VIR0
10
20
30
40
50
****
ns
% o
f C
D3
00
a+
B c
ells
HD HIV-AVIR HIV-VIR0
20
40
60 *****
ns
% o
f C
D3
00
a+
ce
llsin
th
e C
D2
1-
su
bs
et
HD HIV-AVIR HIV-VIR0
10
20
30
40
50
***
ns
% o
f C
D3
00
a+
ce
llsin
th
e C
D2
1+
su
bs
et
Decreased frequency of CD300a+ B cells in HIV infected patients
Silva et al., 2011. Blood
0 103
104
105
<FITC-A>: CD21
0
103
104
105
<P
E-A
>: C
D30
0a
4.9 25
664
0 103 104 105
<FITC-A>: CD21
0
103
104
105
<P
E-A
>: C
D30
0a
1.9 6.6
6922
0 103
104
105
<FITC-A>: CD21
0
103
104
105
<P
E-A
>: C
D30
0a
6.3 3.6
4248
CD21
CD
300a
HD HIV-AVIR HIV-VIR
Deregulation of CD300a Expression on B Cells of HIV Infected Patients
Silva et al., 2011. Blood
Decreased CD300a Expression on Circulating Mature B Cells From HIV Infected Patients
HD HIV-AVIR HIV-VIR0
2000
4000
6000
8000
ns**
*
Plasma blasts
CD
300a
MF
I
HD HIV-AVIR HIV-VIR0
500
1000
1500
*****
ns
Resting Memory
HD HIV-AVIR HIV-VIR0
200
400
600
800
******
ns
Activated Memory
HD HIV-AVIR HIV-VIR0
100
200
300
400
500 *****
ns
Atypical Memory (exhausted)
CD
300a
MF
I
HD HIV-AVIR HIV-VIR0
100
200
300
400
*ns
ns
Naive
Silva et al., 2011. Blood
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
Impaired binding of CD300a-Ig Q94, a SNP linked to psoriasis susceptibility
Simhadri et al., 2012. Blood
Potential roles of CD300 molecules
Immature DC Mature DC
Gasiorowski et al. 2013. Immunol Letters
The immuno-modulatory role of CD300a
Naïve/resting Immune System
Activated Immune System
Stimulating factors:Antigen recognitionDanger signalsInflammatory milieuOncogenic Transformation
Eradication of InsultCytotoxicityPro-inflammatorycytokines
Shut Down
Removal of Activated Cells:Apoptosis and subsequent phagocytosis (Macrophages).Cytolysis (NK cells).
Activated cells express ligands for receptors expressed on NK cells
and macrophages.
CD300a
Inhib
itory
Functi
on Modulatory function bycreating anti-inflammatoryenvironment
Acknowledgements
NIAID-RCBS• Rodolfo Silva• Seung Choi• Linjie Tian • John E. Coligan
Borrego Lab Venkateswara Simhadri Karen Debell John Mariano Qing Zhou Aleksandra Gil-Krzewska Milena Dimitrova
NIAID-LIR Susan Moir Lela Kardava
University of Sevilla Manuel Leal Sara Ferrando-Martínez
University of Córdoba José Peña