Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence suggests that they activate dendritic cells (DCs) via Toll-like receptors (TLRs)1,2. For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed3, activates DCs via multiple TLRs to stimulate pro-inflammatory cytokines4,5. Triggering specific combinations of TLRs in DCs can induce synergistic production of cytokines6, which results in enhanced T cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that programs such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus Toll-like receptor (TLR) ligands 4 + 7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with a single TLR ligand. Consistent with this there was enhanced persistence of germinal centers (GCs), and of plasma cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma cell response, relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated early programming towards B cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells (DCs), as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

The generation and selection of somatic antibody mutants are key elements of acquired immunity, essential for the affinity maturation of antibody responses dependent on T cells. The mutants are generated through a mechanism that introduces point mutations at high rate into rearranged variable (V) region genes in the course of cell proliferation. Their appearance coincides with the generation of germinal centres, which are characterized by oligoclonal B-cell proliferation and have been suggested to be the microenvironment in which antibody mutants are generated. We report here direct evidence for this hypothesis. Rearranged V-region genes were amplified from the genomic DNA of cells picked from individual germinal centres. The sequence analysis of these genes revealed that most represent cells of distinct B-cell clones which expanded locally, generating somatic antibody mutants at high rate. By contrast, antigen-induced proliferation of B cells at another site, periarteriolar lymphocyte sheath-associated foci, was not associated with somatic hypermutation.

SUMMARY During acute infections, a small population of effector CD8 T cells evades terminal differentiation and survives as long-lived memory T cells. We demonstrate that the transcriptional repressor Blimp-1 enhances the formation of terminally differentiated CD8 T cells during LCMV infection, and Blimp-1 deficiency promotes the acquisition of memory cell properties by effector cells. Blimp-1 expression is preferentially increased in terminally differentiated effector and “effector memory” (TEM) CD8 T cells, and gradually decays after infection as central memory (TCM) cells develop. Blimp-1-/- effector CD8 T cells show some reduction in effector molecule expression, but primarily develop into memory precursor cells that survive better, and more rapidly acquire several TCM attributes, including CD62L and IL-2 expression and enhanced proliferative responses. These results reveal a critical role for Blimp-1 in controlling terminal differentiation and suppressing memory cell developmental potential in effector CD8 T cells during viral infection.

SummaryAfter primary immunization with an immunogenic conjugate of(4-hydro)cy-3-nitrophenyl)acetyl, two anatomically and phenotypically distinct populations of antibody-forming cells arise in the spleen. As early as 2 d after immunization, foci of antigen-binding B cells are observed along the periphery of the periarteriolar lymphoid sheaths. These foci expand, occupying as much as 1% of the splenic volume by day 8 of the response . Later, foci grow smaller and are virtually absent from the spleen by day 14. A second responding population, germinal center B cells, appear on day 8-10 and persist at least until day 16 post-immunization . Individual foci and germinal centers represent discrete pauciclonal populations that apparently undergo somatic evolution in the course of the primary response. We suggest that foci may represent regions of predominantly interclonal competition for antigen among unmutated B cells, while germinal centers are sites of intraclonal clonal competition between mutated sister lymphocytes . efore the selective action ofantigens, the antibody repertoire of adult mice appears to be determined by a collection of stochastic processes. Probabilistic use and association ofV, D, and J

SummaryIn the murine spleen, germinal centers are the anatomic sites for antigen-driven hypermutation and selection of immunoglobulin (Ig) genes. To detail the kinetics of Ig mutation and selection, 178 VDJ sequences from 16 antigen-induced germinal centers were analyzed. Although germinal centers appeared by day 4, mutation was not observed in germinal center B cells until day 8 postimmunization; thereafter, point mutations favoring asymmetrical transversions accumulated until day 14. During this period, strong phenotypic selection on the mutant B lymphocytes was inferred from progressively biased distributions of mutations within the Ig variable region, the loss of crippling mutations, decreased relative clonal diversity, and increasingly restricted use of canonical gene segments. The period of most intense selection on germinal center B cell populations preceded significant levels of mutation and may represent a physiologically determined restriction on B ceUs permitted to enter the memory pathway. Noncanonical Ig genes recovered from germinal centers were mostly unmutated although they probably came from antigen-reactive cells. Together, these observations demonstrate that the germinal center microenvironment is rich and temporally complex but may not be constitutive for somatic hypermutation. ,•ter immunization with immunogenic conjugates of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP), 1 Ig hypermutation and selection take place in germinal centers (GC) (1), specialized histologic structures of secondary lymphoid tissues (2-4). Splenic GC support oligodonal B cell populations (2, 4, 5) that result from colonizing migration by antigen-activated (6) lymphocytes from the region of the periarteriolar lymphoid sheath (PALS) (4, 6, 7). Nonmigrating sister cells remain associated with the PALS, establishing large loci of antibody-forming cells (4, 6, 7). Interestingly, although focus and GC B cell populations arise from common founders, Ig hypermutation is not evident in loci (1, 6) and presumably is dependent upon the GC microenvironment.Despite intensive study, our understanding of somatic Ig mutation and selection remains elementary. Mutations are introduced into rearranged V regions of transcriptionally active Igh-and -I loci (8, 9) at the rate of 10 -3 mutations/bp per cell/generation (10). Mutations are predominately single nucleotide substitutions (90-95%) although small deletions 1 Abbreviations used in this paper: CG, chicken gamma globulin; FW, framework region; GC, germinal center; NP, (4-hydroxy-3-nitrophenyl)-acetyl; PALS, periarteriolar lymphoid sheath; PNA, peanut agglutinin. and insertions also have been noted (11-15). These mutations are distributed asymmetrically within an "~2-kbp region whose 5' boundary is defined by the V. promoter sequence (15, 16); the focus of this distribution is the rearranged V(D)J elements (15). Although mutation is believed to be approximately random (11-14), strand biases (17) and mutational hot spots (11, 18) have been reported. The mechanism oflg hypermutation i...