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)