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EXPLORING NEW MECHANISMS TO UNDERSTAND THE
COMPLEX B CELL DYSREGULATION PRESENT IN COMMON
VARIABLE IMMUNODEFICIENCY: IMPAIRED CPG
DEMETHYLATION ASSOCIATES WITH B CELL PHENOTYPE AND
PROLIFERATION RATE
Lucía del Pino Molina, PhD
Immunology Department. La Paz University Hospital,
Lymphocyte Pathophysiology in Immunodeficiencies group, IdiPAZ
del Pino-Molina L, Rodríguez-Ubreva J, Torres Canizales J, Coronel-Díaz M, Kulis M, Martín-
Subero JI, van der Burg M, Ballestar E, López-Granados E.
Front Immunol. 2019 Apr 24;10:878. doi: 10.3389/fimmu.2019.00878. eCollection 2019.
PIDs
https://esid.org/Working-Parties/Registry-Working-Party/ESID-Database-Statistics
Common Variable Immunodeficiency patients
Otitis Sinusitis
Upper respiratory
infections
Lower respiratory
infections
Pneumonia
Infections
Splenomegaly
Bronchiectasias
Gastrointestinal
complications
Autoimmune
thombocytopenia
Stomach cancerdiarrhea
Low serum
immunoglobulins
Low vaccination
response
Lymphoproliferation
Low memory
B cells
• Variability in age of onset
• In some cases progression:
• Can CVID arise only as a consequence of DNA sequence variations?
isolated IgA deficiency
Low IgA levels
CVID
Low IgA levels Low IgG levels
Bogaert DJA, et al. Med. Genet. 2016 Sep;53(9):575-90
Common Variable Immunodeficiency Disorders
• Variability in age of onset
• In some cases progression:
• Can CVID arise only as a consequence of DNA sequence variations?
• Heterogeneous disease
• CVID uncommon epidemiology and complex pathogenesis
• CVID disorders needs from new approaches
isolated IgA deficiency
Low IgA levels
CVID
Low IgA levels Low IgG levels
Common Variable Immunodeficiency Disorders
• Variability in age of onset
• In some cases progression:
• Can CVID arise only as a consequence of DNA sequence variations?
• CVID uncommon epidemiology and complex pathogenesis
• CVID disorders needs from new approaches
isolated IgA deficiency
Low IgA levels
CVID
Low IgA levels Low IgG levels
Epigenetic approach
Hypothesis: “Epigenetic dysregulation
might contribute to CVID pathology”
Common Variable Immunodeficiency Disorders
Epigenetics
Goldberg et al. Cell 2007 Feb 23;128(4):635-8.)
Heritable changes in the chromatin structure
that modify gene expression without changes in
the DNA sequence
DNA methylation
-Addition of methyl group in cytosines at CpG dinucleotides
-Mediated by DNMTs
-CpG dinucleotides are clustered in CpG islands near gene
promoters,
interfering with accesibility and recruitment of
transcription factors
-Demethylation is induced by proliferation or by TET family
enzymes.
Martin-Subero JI, Oakes CC. Semin Cancer Biol. 2018 Aug;51:139-148.
Epigenetic mechanism control transcriptional
programs during B cell differentiation
Transcription factors + epigenetic marks
regulate the expression of genes
related to B-cell functions
Plasma cellSerum
immunoglobulins
HSC Pro B Pre B inmature
Bone marrow
Naive B cell
Unswitched
memory B cell
Germinal
center
Switched
memory B cell
Secondary
lymphoid organs
DNA methylome is
modified during B
cell differentiation,
affecting several
million CpG sites Zouali, M. The epigenetic
landscape of B lymphocyte
tolerance to self. FEBS Lett.
(2013)
B cell development
Plasma cellSerum
immunoglobulins
HSC Pro B Pre B immature
Bone marrow
Naive B cell
Unswitched
memory B cell
Germinal
center
Switched
memory B cell
Secondary
lymphoid organs
From Naive to memory B cell
GC reaction
Massive methylome reconfiguration
High replication rate
DNA methylation
B cell development
Plasma cellSerum
immunoglobulins
HSC Pro B Pre B immature
Bone marrow
Naive B cell
Unswitched
memory B cell
Germinal
center
Switched
memory B cell
Secondary
lymphoid organs
Memory B cells
epigenetically prepared
rapidly differentiate into
plasma cells
Epigenetic mechanisms confer memory B cells the
ability to undergo rapid and dramatic gene expression
changes by poising its genome through DNA
methylation.
CVID blocks in B cell development
Plasma cellSerum
immunoglobulins
HSC Pro B Pre B immature
Bone marrow
Naive B cell
Unswitched
memory B cell
Germinal
center
Switched
memory B cell
Secondary
lymphoid organs
CVID present impaired B cell differentiation
We decided to analyze DNA
methylation in B cell subsets
Sorting B
subpopulations
22 CVID
17 Healthy
donors
Bisulfite
modification
DNA
extractionPCR amplification
Pyrosequencing
Genes implicated in B cell differentiation,
activation
DNA methylation study Methods
Healthy donors displayed demethylation from naïve to memory B cells in selected CpGs
Naïve B cells from CVID patients have similar methylation levels than controls
Unswitched and switched memory B cells from CVID patients higher methylation levels
than controls for some of the selected CpGs
Unswitched and switched memory B cells from CVID patients higher methylation levels
than controls for some of the selected CpGs
Normal numbers memory
B cells
Intensity of impairment in DNA demethylation levels associated with B cell phenotype
HD
CVID normal B cell phenotype
CVID reduced switched B cells
Reduced only switched memory B cells
Intensity of impairment in DNA demethylation levels associated with B cell phenotype
Reduced unswitched and
switched memory B cells
Intensity of impairment in DNA demethylation levels associated with B cell phenotype
Demethylation could be associated with cell divisions
Evaluation of B cell replication history
Impaired CpG
demethylation observed in
CVID switched memory B
cells could be related to the
proliferation rate?
Evaluation of B cell replication history
Impaired CpG
demethylation observed in
CVID switched memory B
cells could be related to the
proliferation rate?
No correlation in HD, neither
for most of CpG studied in
CVID, but...
AID negative correlation DNA methylation and cell divisions
AID negative correlation DNA methylation and cell divisions
Reduced SHM in CVID Memory B Cells with hypermethylated AICDA
% S
HM
0
1 0
2 0
3 0
4 0
*** ***P-value=0,0004 P-value=0,0001
HDCVID
Naive USm Sm
in some
relevant
genes for B
cell function
Our study reinforce the hypothesis of altered
demethylation during B cell differentiation as a
contributing pathogenic mechanism to the impairment of
B cell function and maturation in CVID.
In memoria
Dr. Margarita Salas
died yesterday
A referent for the
Spanish Science
A pioneer in molecular
biology
Collaborators:
Esteban Ballestar, Fundación Josep Carreras, Barcelona, Spain
Javier R. Ubreva, Fundación Josep Carreras, Barcelona, Spain
Mirjam van der Burg, Leiden University Medical Center, Netherlands
José I. Martín-Subero, IDIBAPS Barcelona, Spain
Marta Kulis Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain
Lucía del Pino Molina, PhD
Eduardo López Granados, MD, PhD
Juan Torres, MD
María Coronel
Immunology department,
La Paz University Hospital:
Rebeca Rodríguez
Carmen Cámara
Carla Gianelli
Yadira Bravo
Elena Sánchez
Ana Martínez
Pilar Nozal
Miguel González
María Bravo
Noelia Carballeda
Borja Hernández
Argentina Colmenero
Juan Valdivieso
Elisabeth Matas
Andrea González
We thank all the patients and families
Support:
FIS PI16/01605 (Fondo de Investigación Sanitaria Instituto de Salud Carlos III, Madrid, Spain)