Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

SummaryUsing a series of phenotypic markers that include immunoglobulin (Ig)D, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing different stages of B cell differentiation that included slgD + (Bin1 and Bm2), germinal center (Bin3 and Bin4), and memory (BinS) B cells. To establish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction and subsequent sequence analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest V. families (VH4, V.5, and VH6) in order to facilitate the mutational analysis. In agreement with previous reports, we found that the somatic mutation machinery is activated only after B cells reach the germinal center and become centroblasts (Bm3). Whereas 47 independently rearranged IgM transcripts from the Bml and Bm2 subsets were nearly germline encoded, 57 Bm3-, and Bm4-, and BmSderived IgM transcripts had accumulated an average of 5.7 point mutations within the V. gene segment. 3' transcripts corresponding to the same V. gene families were isolated from subsets Bm3, Bin4, and Bm5, and had accumulated an average of 9.5 somatic mutations. We conclude that the molecular events underlying the process of somatic mutation takes place during the transition from IgD +, CD23 + B cells (Bm2) to the IgD-, CD23-, germinal center centroblast (Bm3). Furthermore, the analysis of Ig variable region transcripts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. The availability of these subpopulations should permit the identification of the functional molecules relevant to each stage of 13 cell differentiation.T he variable regions of the two critical antigen receptors of the immune system, the T cell receptor and the immunoglobulin molecule, are encoded by five different genetic elements that, in the germline, are separated by thousands of base pairs (1, 2). A recombination machinery shared by T and B cells brings these elements together into functional TCR V~/V~ and Ig V./V~ chains (3). Availability of a broad array of germline genes, generation of random amino acids during the process of rearrangement, and combinatorial association of Vo/V~ and VH/V~ chains are essential steps in the generation of diversity within the T and B cell repertoires. B cells display the unique property of accumulating somatic mutations in their Ig variable region genes, further contributing to increase the almost limitless number of antigenic specificities (1).Although a large body of information has accumulated in recent years concerning the repertoire of human Ig variable region genes, both at the level of genomic organization and expression, our current knowledge about the mechanism of somatic mutation remains elementary. Mutations are introduced only into rear...

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with 'non-self' components (LPS) and 'self' components (apoptotic cells) produce distinct macrophage responses.

All human and murine immunoglobulin heavy chain variable region (VH) genes contain the sequence ATGCAAAT approximately 70 nucleotides 5' from the site of transcription initiation. This octanucleotide, in reverse orientation, is also found in all light chain variable region (VL) genes, and in the immunoglobulin heavy chain transcriptional enhancer. Transfection studies have established that this octamer is involved in the lymphoid-specific transcription of immunoglobulin genes. Octamer-containing fragments have been reported to bind a factor present in nuclear extracts of human cell lines; however, identical binding activity was detected in both B lymphoid and non-lymphoid cells. Here we establish that nuclear extracts from distinct cell types differ in their ability to interact with octamer-containing fragments. We have also detected a DNA-protein interaction that may be involved in the cell-type specificity of immunoglobulin expression, and we have determined that a sequence upstream of the octamer participates in an interaction with a nuclear protein(s).

Contrary to the general precepts of the clonal selection theory, several recent studies have provided evidence for the secondary rearrangement of immunoglobulin (Ig) genes in peripheral lymphoid tissues. These analyses typically used transgenic mouse models and have only detected secondary recombination of Ig light chain genes. Although Ig heavy chain variable region (VH) genes encode a substantial element of antibody combining site specificity, there is scant evidence for VH gene rearrangement in the periphery, leaving the physiological importance of peripheral recombination questionable. The extensive somatic mutations and clonality of the IgD+Strictly-IgM−CD38+ human tonsillar B cell subpopulation have now allowed detection of the first clear examples of receptor revision of human VH genes. The revised VDJ genes contain “hybrid” VH gene segments consisting of portions from two separate germline VH genes, a phenomenon previously only detected due to the pressures of a transgenic system.