Final Report Summary - NEONATAL B CELLS (Functional analysis of the neonatal B cell compartment)
Neonatal life is characterized by heightened sensitivity to infectious agents. The sensitivity of newborns to infectious diseases is caused by an immaturity of the immune system which could be due to either qualitative or quantitative differences between neonatal and adult immune cells. With respect to the B cell compartment, several differences have been observed between adults and neonates. In peripheral blood, phenotypical and functional different B cell subpopulations can be identified including transitional 1 (T1) and 2 (T2), naïve and memory B cells. In contrast to adult blood, cord blood contains an increased number of transitional B cells whereas memory B cells are almost completely absent.
This project was designed to investigate and better understand functional differences between the human neonatal and adult B cell compartment. One key question is whether these differences are merely caused by a different composition of B cell subpopulations or whether, in addition, single B cell subsets are intrinsically immature, resulting in overall altered function. Therefore, we wanted to test the hypothesis if neonatal B cells are intrinsically immature and defective in comparison to adult B cells.
For all experiments, B cells were purified from buffy coats or fresh blood (for live assays) obtained from healthy individuals or from cord blood. No obvious differences in surface expression were found with respect to CD22, CD10, MHC class II and CD86. Only IgM was significantly higher expressed on neonatal T1 und T2 B cells. In respect to the BAFF system, we determined surface expression of the BAFF receptors BAFF-R, TACI and BCMA. However, no significant differences were detected between neonatal versus adult B cell subpopulations.
Proliferation was analysed in response to stimulation via Toll-like receptors (TLR) ligands or the B cell receptor (BCR) with or without the addition of several cytokines. The data show reduced proliferation of neonatal T2 and naïve B cells in response to CpG +/- anti-IgG/IgM/IgA but enhanced proliferation of neonatal T1, T2 and naïve B cells in response to stimulation with CpG alone.
Cell cycle analysis revealed that neonatal B cell subpopulations (T2 and naïve B cells) are to a higher percentage found in G1 phase and are therefore more prone to enter the cell. KREC (kappa-deleting recombination excision circle) were determined as a measure for proliferation in vivo but no differences could be detected.
Next, production of immunoglobulins was analysed. Most interestingly, our results demonstrate that neonatal B cells only produce IgM and are not capable to class-switch to IgG or IgA. Analysis of cytokine secretion revealed higher secretion of IL-6 but decreased production IL-10 by neonatal B cell subpopulations after stimulation.
Investigation of proximal signalling by phosflow assays showed significantly enhanced phosphorylation of tyrosine residues in neonatal compared to adult T1 and partially T2 B cells but no differences in AKT activation. In addition, Ca2+ flux was higher in neonatal B cell subpopulations after stimulation with anti-IgM as compared to their adult counterparts.
In summary, our results indicate that neonatal B cells are intrinsically immature and altered in comparison to adult B cells. We are now beginning to analyse the mechanism behind this finding. In particular, we are looking at epigenetic changes (microRNA, Methylation)
The results of this proposal contribute to a better understanding of the immune system in early life and the mechanisms that account for differences in the B cell compartment between adults and neonates. This knowledge will lead to a significant improvement in the therapy and prevention of infections not only in newborns but also in preterm babies.
This project was designed to investigate and better understand functional differences between the human neonatal and adult B cell compartment. One key question is whether these differences are merely caused by a different composition of B cell subpopulations or whether, in addition, single B cell subsets are intrinsically immature, resulting in overall altered function. Therefore, we wanted to test the hypothesis if neonatal B cells are intrinsically immature and defective in comparison to adult B cells.
For all experiments, B cells were purified from buffy coats or fresh blood (for live assays) obtained from healthy individuals or from cord blood. No obvious differences in surface expression were found with respect to CD22, CD10, MHC class II and CD86. Only IgM was significantly higher expressed on neonatal T1 und T2 B cells. In respect to the BAFF system, we determined surface expression of the BAFF receptors BAFF-R, TACI and BCMA. However, no significant differences were detected between neonatal versus adult B cell subpopulations.
Proliferation was analysed in response to stimulation via Toll-like receptors (TLR) ligands or the B cell receptor (BCR) with or without the addition of several cytokines. The data show reduced proliferation of neonatal T2 and naïve B cells in response to CpG +/- anti-IgG/IgM/IgA but enhanced proliferation of neonatal T1, T2 and naïve B cells in response to stimulation with CpG alone.
Cell cycle analysis revealed that neonatal B cell subpopulations (T2 and naïve B cells) are to a higher percentage found in G1 phase and are therefore more prone to enter the cell. KREC (kappa-deleting recombination excision circle) were determined as a measure for proliferation in vivo but no differences could be detected.
Next, production of immunoglobulins was analysed. Most interestingly, our results demonstrate that neonatal B cells only produce IgM and are not capable to class-switch to IgG or IgA. Analysis of cytokine secretion revealed higher secretion of IL-6 but decreased production IL-10 by neonatal B cell subpopulations after stimulation.
Investigation of proximal signalling by phosflow assays showed significantly enhanced phosphorylation of tyrosine residues in neonatal compared to adult T1 and partially T2 B cells but no differences in AKT activation. In addition, Ca2+ flux was higher in neonatal B cell subpopulations after stimulation with anti-IgM as compared to their adult counterparts.
In summary, our results indicate that neonatal B cells are intrinsically immature and altered in comparison to adult B cells. We are now beginning to analyse the mechanism behind this finding. In particular, we are looking at epigenetic changes (microRNA, Methylation)
The results of this proposal contribute to a better understanding of the immune system in early life and the mechanisms that account for differences in the B cell compartment between adults and neonates. This knowledge will lead to a significant improvement in the therapy and prevention of infections not only in newborns but also in preterm babies.