Human memory B cells (MBCs) are generated and diversified in secondary lymphoid tissues throughout the organism. A paired immunoglobulin (Ig)-gene repertoire analysis of peripheral blood (PB) and splenic MBCs from infant, adult, and elderly humans revealed that throughout life, circulating MBCs are comprehensively archived in the spleen. Archive MBC clones are systematically preserved and uncoupled from class-switching. Clonality in the spleen increases steadily, but boosts at midlife, thereby outcompeting small clones. The splenic marginal zone (sMZ) represents a primed MBC compartment, generated from a stochastic exchange within the archive memory pool. This is supported by functional assays, showing that PB and splenic CD21+ MBCs acquire transient CD21high expression upon NOTCH2-stimulation. Our study provides insight that the human MBC system in PB and spleen is composed of three interwoven compartments: the dynamic relationship of circulating, archive, and its subset of primed (sMZ) memory changes with age, thereby contributing to immune aging.

Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

SUMMARYThe human infant B cell system is considered premature or impaired. Here we show that neonates have mature and fully functional B cell subsets as seen in adults, albeit with distinct transcriptional programs providing accelerated responsiveness to T cell independent and T cell dependent stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into antibody secreting cells, thereby presumably limiting memory B cell formation. The neonatal Ig-repertoire is highly variable, but conserved, showing 8% recurrent B cell receptor (BCR) clonotypes shared between neonates. Our study demonstrates that neonatal B cells are neither premature nor impaired, but differ from their adult counterpart in a conserved BCR repertoire and rapid but transient response dynamics. These properties may account for the sensitivity of neonates to infections and limited effectivity of vaccination strategies. Finally, our findings have implications for a limited comparability of mouse models to human infant B cell immunity.

While quantitative proteomics is a key technology in biological research, the routine industry and diagnostics application is so far still limited by a moderate throughput, data consistency and robustness. In part, the restrictions emerge in the proteomics dependency on nanolitre/minute flow rate chromatography that enables a high sensitivity, but is difficult to handle on large sample series, and on the stochastic nature in datadependent acquisition strategies. We here establish and benchmark a labelfree, quantitative proteomics platform that uses microlitre/minute flow rate chromatography in combination with dataindependent SWATH acquisition. Being able to largely compensate for the loss of sensitivity by exploiting the analytical capacities of microflow chromatography, we show that microLCSWATHMS is able to precisely quantify up to 4000 proteins in an hour or less, enables the consistent processing of sample series in highthroughput, and gains quantification precisions comparable to targeted proteomic assays. MicroLCSWATHMS can hence routinely process hundreds to thousands of samples to systematically create precise, label free quantitative proteomes.

Young children and older adults suffer from enhanced susceptibility to infections with blood-borne pathogens. An essential step towards immunity is the establishment of a splenic marginal zone (sMZ), which is immature at below 2 years of age. At approximately 5 years of age, an adult level of protection is reached but wanes again in older adults. Although the infant sMZ is thought to contain mostly naïve B cells, memory B cells are recruited to and recirculate from the sMZ throughout life, and class-switched sMZ B cells dominate in older adults. For a better resolution of naïve versus memory B-cell subset accumulation in the sMZ, we performed a single cell-based gene expression analysis of (CD21highIgMhigh) sMZ B cells among five healthy donors (age 3 to 48 years) and validated the sMZ B-cell subset composition by flow cytometry of 147 spleen biopsies (age 0 to 82 years). We identified a major sMZ B-cell subpopulation, which is abundant at birth but decreases with age. These cells lack CD27 expression but carry a weak-to-intermediate memory B-cell signature. These CD27neg sMZ B cells are either IGHV-unmutated or carry only a few IGHV mutations early in life but show average memory B-cell IGHV mutation frequencies (>3%) in adults. The activation and proliferation potential of CD27neg sMZ B cells is significantly above that of non-sMZ B cells already in children. Our study suggests that the human sMZ B-cell pool changes with age, encompassing a major population of lowly Ig-mutated CD27neg but antigen-experienced B cells early in life.

Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV mutation phylogenetics.

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