Lucía Del Pino Molina, Spain

Foundation for Biomedical Research La Paz University Hospital Clinical Immunology

Presenter Of 2 Presentations

E-Poster Discussion B Cell Biology

IMPAIRED CPG DEMETHYLATION IN COMMON VARIABLE IMMUNODEFICIENCY ASSOCIATES WITH B CELL PHENOTYPE AND PROLIFERATION RATE

Lecture Time
13:22 - 13:29
Room
Station 1
Date
20.09.2019, Friday
Session Time
13:15 - 14:20
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach.

Methods

We selected CpG sites for analysis undergo significant demethylation from naïve to memory B cells in controls. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and controls. In the same samples we analyzed the replication history by the k-deleting recombination excision circle (KREC) assay. And the somatic hypermutation using the Igk-restriction enzyme hot spot mutation assay.

Results

We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signalling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate.

Conclusions

Our new results 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 particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.

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Poster Display B Cell Biology

NOVEL FLOW CYTOMETRIC TOOLS FOR THE DISSECTION OF THE PRE-GERMINAL CENTER B CELL MATURATION PATHWAY IN COMMON VARIABLE IMMUNODEFICIENCY

Lecture Time
10:01 - 10:02
Room
Poster Area
Date
19.09.2019, Thursday
Session Time
10:00 - 17:00
Board Number
11
Presentation Topic
B Cell Biology

Abstract

Background and Aims

Common Variable Immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency. A significant proportion of patients present decreased number of circulating B cells, and defective or deregulated pre-germinal center B cell compartment.

We aim to precisely trace the maturation pathway of the pre-germinal center compartment in CVID patients, to identify if a deregulated or derailed maturation could already indicate an impaired early development in bone marrow, or suggest alterations in peripheral B cell homeostasis in a proportion of patients.

Methods

A total of 100 CVID patients and 56 HD were studied with standardized protocols within the collaborative and multicenter frame of the PID-group of the EuroFlow consortium. Patients samples and clinical data were collected at 7 different sites.

The distribution of distinct B-cell subsets were analyzed by flow cytometry, with the EuroFlow 8-color pre GC B-cell tube. CD19+ B-cells were subclassified into 11 different subpopulations.

Results

We created a normal maturation B cell pathway focused in the pre GC B cell compartment of HD with the maturation tool of the Infinicyt software. We performed the same analysis to a cohort of 100 CVID patients and plotted the individual results to the normal, in order to detect aberrant populations in preGC in CVID that may have followed a diverse maturation pathway, and identify aberrant expression of markers in CVID patients.

Conclusions

We sought for correlations among alterations in the preGC compartment (absolute counts and expression markers) with clinical data records looking for potential implications of phenotypic aberrancies and clinical complications.

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