Amniotic fluid contains fetal immature B-lymphocytes capable to give rise in vitro to fully competent B cells

LORENA JURIOL; FEDERICO JENSEN; IMRE BOMMER; MAREK ZYGMUNT; NATALIN VALEFF; DAMIAN MUZZIO

Mar del Plata

Congreso; LXI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC) LXIV REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI) XLVIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE FARMACOLOGÍA EXPERIMENTAL (SAFE) VII REUNIÓN ANUAL DE LA SOCIEDAD; 2016

Albeit diverse components of the innate immunity have been found in amniotic fluid (AF), the presence of B-cells was not investigated so far. B-lymphocytes are classified in B1 and B2 B cells. While B2 B cells are continuously generated through the post-natal life, B1 B cells are mainly originated during embryonic life from precursors (CD19+B220-) present in the yolk sac and fetal liver. The aim of this work was to make a full characterization of the B-cells found in the AF.C57BL/6 (H2b) females were mated with BALB/c (H2d) males and sacrificed at day 14 of pregnancy. B cells were magnetically isolated from AF. CD19+ isolated B cells were further stained with specific antibodies against B220, CD93, IgM and H2d for phenotypic characterization using flow cytometry. To analyze the maturation capacity, activation and cytokines/antibodies production, AF isolated CD19+ B cells were co-cultured with a bone marrow stromal cell line in the presence of IL-7 and Flt-3 for 10 days and the phenotype was analyzed again. Additionally, some cells were further stimulated for an extra 24 h with LPS. PMA/ionomycin were added during the last 5 h of culture. Levels of cytokines and immunoglobulin were measured in the supernatants.Phenotypic analysis of AF isolated CD19+ cells permitted the identification of two populations of fetal B cells: an immature population (CD19+B220-IgM-, ≈35%) and a more mature population (CD19+B220+IgM+, ≈45%). Importantly, all CD19+ B cells expressed H2d denoting fetal origin. After 10 days in culture the majority of CD19+ B cells co-expressed B220 and IgM while down-regulated the immature marker CD93. Besides, stimulated AF B cells produced TNFα and IL-10. Interestingly, both, LPS stimulated and non-stimulated B cells, spontaneously produced IgM. We demonstrated here the existence of fetal immature B cells in the AF that are fully competent to continue their development and produce antibodies spontaneously as well as cytokines upon activation.