Highly enriched germinal center (GC) B cell populations are essential for studying humoral immunity. Current MACS protocols that isolate untouched GC B cells require GC induction and typically require further FACS purification with direct antibody labeling to achieve sufficiently high purities. We present a MACS protocol with progressive and repeated negative selections that yields highly purified untouched GC B cells from both unimmunized and GC-induced mice and allows further FACS isolation of unlabeled GC B cells from remaining debris by scatter.
© 2022 The Author(s).

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30-40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data.

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)-translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies.

Vaccines for the prophylactic and/or therapeutic immunization against hepatotropic pathogens (e.g., hepatitis B and hepatitis C virus) should establish long-lasting, specific antiviral effector/memory CD8+ T cell immunity in the liver. We describe a novel peptide-based vaccine in which antigenic major histocompatibility complex Class I-binding peptides are fused to a cationic (e.g., human immunodeficiency virus tat-derived) domain and complexed to immune-stimulating oligonucleotides. This vaccine formulation efficiently primes liver-homing, Class I-restricted CD8+ effector/memory T cell responses. In different antigen systems, this formulation was more potent in priming liver-homing CD8+ T cell responses than DNA-based vaccines delivering the same epitopes. CD8+ T cell priming was independent of CD4+ T cell "help" but submitted to regulatory control by CD25+ CD4+ T cells. The vaccine efficiently primed memory/effector CD8+ T cells detectable in the liver for more than 3 months after a single injection. With increasing time after priming, the phenotype of these specific memory CD8+ T cells shifted from an effector memory to a central memory type. The vaccine could override T cell tolerance in mice expressing the relevant antigen from a transgene in the liver. The CD8+ T cell immunity in the liver primed by this peptide formulation could be boosted by challenge injections. In conclusion, we describe a simple and potent vaccine formulation that has the potential to generate or reconstitute specific CD8+ T cell immunity to hepatotropic pathogens in the liver.
Copyright 2004 American Association for the Study of Liver Diseases