Secondary Immunization Generates Clonally Related Antigen-Specific Plasma Cells and Memory B Cells

Abstract

Rechallenge with T cell-dependent Ags induces memory B cells to re-enter germinal centers (GCs) and undergo further expansion and differentiation into plasma cells (PCs) and secondary memory B cells. It is currently not known whether the expanded population of memory B cells and PCs generated in secondary GCs are clonally related, nor has the extent of proliferation and somatic hypermutation of their precursors been delineated. In this study, after secondary tetanus toxoid (TT) immunization, TT-specific PCs increased 17- to 80-fold on days 6–7, whereas TT-specific memory B cells peaked (delayed) on day 14 with a 2- to 22-fold increase. Molecular analyses of VHDJH rearrangements of individual cells revealed no major differences of gene usage and CDR3 length between TT-specific PCs and memory B cells, and both contained extensive evidence of somatic hypermutation with a pattern consistent with GC reactions. This analysis identified clonally related TT-specific memory B cells and PCs. Within clusters of clonally related cells, sequences shared a number of mutations but also could contain additional base pair changes. The data indicate that although following secondary immunization PCs can derive from memory B cells without further somatic hypermutation, in some circumstances, likely within GC reactions, asymmetric mutation can occur. These results suggest that after the fate decision to differentiate into secondary memory B cells or PCs, some committed precursors continue to proliferate and mutate their VH genes.

Efficient tetanus toxoid immunization on vitamin D supplementation

“…In contrast, the data indicate that induction of TT-specific IgG was marginally more efficient in vitamin D compared with the placebo group (P ¼ 0.04). These data are in line with previous studies (Ivanov et al, 2006;Enioutina et al, 1999Enioutina et al, , 2008Van der Stede et al, 2001) and attributed most likely to induction of specific IgG1, which is the dominant subclass induced by the vaccination used herein (492% of the circulating specific plasmablasts; Frolich et al, 2010). That vitamin D supplementation did not alter the specific Peripheral blood mononuclear cells were activated before and 7 days after a Tet immunization for 24 h in the presence of Tet or SEB, and the secreted cytokines were determined from the supernatants.…”

Tissue Distribution and Dependence of Responsiveness of Human Antigen-Specific Memory B Cells

“…Single-cell sorting, cDNA synthesis, nested PCR, and sequence analysis For single-cell sorting, PBMCs were stained as described before (28 rTT.C-specific (rTT.C + ) PCs of HD1, HD6, HD2 TX , and S1, S2, S4, S5, and S6, respectively, were sorted into 96-well plates using a FACSAria II cell sorter (BD). Each well contained a reaction mix as described elsewhere, and first-strand cDNA was generated (28,29).…”

Identification and characterization of the constituent human serum antibodies elicited by vaccination

“…Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6-7 d and subsequently rapidly decline to nearly undetectable levels (6,7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).…”

“…We elected to analyze the response to booster immunization of the TT vaccine because (i) it elicits a highly effective neutralizing response that is protective toward Clostridium tetani challenge; (ii) the vaccine is highly efficacious, and as a result, no deaths from tetanus intoxication have been reported in the United States for individuals who have completed at least primary immunization (21); (iii) TT has been used as a model for analyzing B-cell development following vaccination in humans (6,22,23); and (iv) although early serological and mAb studies had pointed to the C-terminal fragment of the toxin heavy chain [recombinant TT fragment C (rTT.C)] as the target for antibody-mediated protection (24), the precise mechanism by which antibodies elicited by the vaccine mediate neutralization has remained unclear.…”