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Objective Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoantibody production. As spleen tyrosine kinase (Syk) is pivotal in B cell activation, these experiments aimed to examine the extent to which Syk was abnormally expressed in SLE B cells and the nature of the B cell subset that differently expressed Syk. Methods B cells from healthy donors and SLE patients were analyzed by flow cytometry to assess basal expression of Syk and phosphorylated Syk. B cell subsets expressing higher levels of Syk were found, and their detailed phenotype, in vitro differentiation into plasmablasts/plasma cells, and Syk induction by cytokines were determined. Results Syk expression was higher in CD27+ memory B cells than in naive B cells from SLE patients. However, a significantly increased frequency of CD27− B cells with bright expression of Syk (Syk++) was found in SLE patients. CD27−Syk++ B cells showed enhanced basal expression of p-Syk and stronger Syk phosphorylation upon B cell receptor (BCR) engagement as compared to CD27−Syk+ B cells. CD27−Syk++ B cells were CD38− as well as CD19++, CD20++, and mainly CD21−, with decreased ABCB1 transporter activity. In contrast to CD27−Syk+ B cells, CD27−Syk++ B cells exhibited enhanced differentiation into CD27++ IgG-secreting cells and expressed somatically mutated BCR gene rearrangements. Syk++ B cells were inducible in vitro by stimulation with interferon-γ, lipopolysaccharide, or tumor necrosis factor α. Conclusion SLE patients exhibit an increased frequency of hitherto unknown CD27−Syk++ memory-like B cells, indicating that intracellular Syk density could distinguish CD27− memory B cells from truly naive B cell subsets. Furthermore, the CD27−Syk++ subset is a candidate for a source of increased plasma cells in SLE. [ABSTRACT FROM AUTHOR]

Memory B cells (mBCs) are a key to immunologic memory, yet their distribution within lymphoid organs and the individual role of these for mBC functionality remain largely unknown. This study characterized the distribution and phenotype of human (Agspecific) mBCs in peripheral blood (PB), spleen, tonsil, and bone marrow. We found that the spleen harbors most mBCs, followed by tonsils, BM, and PB, and we detected no major differences in expression of markers associated with higher maturity. Testing the distribution of tetanus toxoid-specific (TT+) mBCs revealed their presence in PB during steady state, yet absolute numbers suggested their largest reservoir in the spleen, followed by tonsils. To explore the role of both tissues in the maintenance of reactive B cell memory, we revaccinated controls and splenectomized and tonsillectomized individuals with TT. All donor groups exhibited comparable emergence of anti-TT IgG, TT+ plasma cells, and TT+ mBCs in the PB, together with similar molecular characteristics of TT+ plasma cells. In summary, human mBCs recirculate through PB and reside in different lymphoid organs that do not reflect different mBC maturity stages. The spleen and tonsil, although harboring the largest number of overall and TT+ mBCs, appear to be dispensable to preserve adequate responsiveness to secondary antigenic challenge. [ABSTRACT FROM AUTHOR]

There are currently limited insights into the progression of human primary humoral immunity despite numerous studies in experimental models. In this study, we analyzed a primary and related secondary parenteral keyhole limpet hemocyanin (KLH) immunization in five human adults. The primary challenge elicited discordant KLH-specific serum and blood effector B cell responses (i.e., dominant serumKLH-specific IgGand IgMlevels versus dominant KLH-specific IgAplasmablast frequencies). Single-cell IgH sequencing revealed early appearance of highly (>15 mutations) mutated circulating KLH-specific plasmablasts 2 wk after primary KLH immunization, with simultaneous KLH-specific plasmablasts carrying non- and low-mutated IgH sequences. The data suggest that the highly mutated cells might originate from cross-reactive memory B cells (mBCs) rather than from the naive B cell repertoire, consistent with previous reported mutation rates and the presence of KLH-reactive mBCs in naive vaccinees prior to immunization. Whereas upon secondary immunization, serum Ab response kinetics and plasmablast mutation loads suggested the exclusive reactivation of KLH-specific mBCs, we, however, detected only little clonal overlap between the peripheral KLH-specific secondary plasmablast IgH repertoire and the primary plasmablast and mBC repertoire, respectively. Our data provide novel mechanistic insights into human humoral immune responses and suggest that primary KLH immunization recruits both naive B cells and cross-reactive mBCs, whereas secondary challenge exclusively recruits from a memory repertoire, with little clonal overlap with the primary response. [ABSTRACT FROM AUTHOR]

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT+ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10-8-10-10 M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3-CD14-CD19+CD27++CD38++CD20-TT+) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation. [ABSTRACT FROM AUTHOR]

Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19+ PC, CD19- PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DRlowKi-67-CD95lowCD28+CD56+/-, with increased BCL2and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19- BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19- PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19+ and CD19- PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection. [ABSTRACT FROM AUTHOR]

Current subunit vaccines are incapable of inducing Ag-specific CD8+ T cell cytotoxicity needed for the defense of certain infections and for therapy of neoplastic diseases. In experimental vaccines, cytotoxic responses can be elicited by targeting of Ag into crosspresenting dendritic cells (DC), but almost all available systems use target molecules also expressed on other cells and thus lack the desired specificity. In the present work, we induced CD8+ T cell cytotoxicity by targeting of Ag to XCR1, a chemokine receptor exclusively expressed on murine and human cross-presenting DC. Targeting of Ag with a mAb or the chemokine ligand XCL1 was highly specific, as determined with XCR1-deficient mice. When applied together with an adjuvant, both vector systems induced a potent cytotoxic response preventing the outgrowth of an inoculated aggressive tumor. By generating a transgenic mouse only expressing the human XCR1 on its cross-presenting DC, we could demonstrate that targeting of Ag using human XCL1 as vector is fully effective in vivo. The specificity and efficiency of XCRl-mediated Ag targeting to cross-presenting DC, combined with its lack of adverse effects, make this system a prime candidate for the development of therapeutic cytotoxic vaccines in humans. [ABSTRACT FROM AUTHOR]

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination. [ABSTRACT FROM AUTHOR]