Abstract: There has been recent progress in understanding the mechanism of somatic hypermutation in germinal centers. By contrast, there is more theory than certainty about the way those cells that acquire heightened affinity for antigen are subsequently selected. Wang and Carter, 2005Wang Y. Carter R.H. Immunity. 2005; 22 (this issue): 749-761Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar highlight this in the current issue of Immunity in a study of germinal center development in mice with impaired CD19 signaling. There has been recent progress in understanding the mechanism of somatic hypermutation in germinal centers. By contrast, there is more theory than certainty about the way those cells that acquire heightened affinity for antigen are subsequently selected. Wang and Carter, 2005Wang Y. Carter R.H. Immunity. 2005; 22 (this issue): 749-761Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar highlight this in the current issue of Immunity in a study of germinal center development in mice with impaired CD19 signaling. During T cell dependent antibody responses, B cells either grow as plasmablasts outside follicles, or, within follicles, form germinal centers (GC). In GC, B cells selectively introduce mutations into their immunoglobulin variable region genes (Jacob et al., 1991Jacob J. Kelsoe G. Rajewsky K. Weiss U. Nature. 1991; 354: 389-392Crossref PubMed Scopus (870) Google Scholar). Cells with augmented affinity for the antigen are then selected by an antigen and T cell-dependent process and leave the GC either as antibody-producing cells or memory cells (MacLennan, 1994MacLennan I.C.M. Annu. Rev. Immunol. 1994; 12: 117-139Crossref PubMed Scopus (1642) Google Scholar). This selection seems likely to require uptake of antigen via the B cells receptor for antigen (BCR) and subsequent presentation to local CD4 T cells. If this is so, the way the encounter with antigen occurs will have a critical influence on selection. Wang and Carter, 2005Wang Y. Carter R.H. Immunity. 2005; 22 (this issue): 749-761Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar describe how GC B cell selection is altered in mice with mutant CD19 that has lost function through disruption of PI3Kinase-binding sites. CD19 is expressed on B cell progenitors as well as immature and mature B cells. On mature B cells, it is linked to the C3d receptor — CD21. The CD19/CD21 complex plays a critical role by adjusting the threshold for antigen-specific B cell activation (Tedder et al., 1997Tedder T.F. Inaoki M. Sato S. Immunity. 1997; 6: 107-118Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Crosslinking CD21 with the BCR greatly reduces the threshold for B cell activation (Dempsey et al., 1996Dempsey P.W. Allison M.E.D. Akkaraju S. Goodnow C.C. Fearon D.T. Science. 1996; 271: 348-350Crossref PubMed Scopus (972) Google Scholar). Conversely CD19/CD21 sequestration with anti-CD19 antibody increases the extent of BCR crosslinking required to activate B cells (Pezzutto et al., 1986Pezzutto, A., Dorken, B., Rabinovitch, P.S., Ledbetter, J.A., Moldenhauer, G., and Clark, E.A. (1986). Leucocyte Typing III, White Cell Differentiation Antigens, 358–360.Google Scholar). Thus, in the context of GC, impaired CD19 function might be expected to affect both the induction of GC and the way GC B cells are selected. The mice with impaired CD19 signaling form GC, but these are markedly smaller in number and size than those of congenic wild type controls. Somatic hypermutation is induced, but the frequency of mutations identified in the immunoglobulin variable region genes of GC B cells of deficient mice was about one fifth normal. Canonical mutations that increase affinity, which are frequently found in wild type GC, were poorly represented in GC of the deficient mice. This seems counter intuitive because BCR signaling is reduced through loss of CD19/CD21 function and this would be expected to increase the minimum BCR affinity required for selection. Perhaps the different repertoire found in the GC of CD19-impaired mice reflects a raised affinity threshold for recruiting B cells into the response. This could be compounded either by a lower rate of proliferation or hypermutation or both of these. The development of GC was also considered in this paper. In common with earlier reports B cell growth in follicles is first apparent within the network of follicular dendritic cells (FDC). Later proliferating cells also appear at one pole of the GC. Conventionally the FDC rich area of the GC is the light zone and the B blasts outside the FDC network occupy the dark zone. A novel observation indicates that the cells in the dark zone have switched from IgM, while those in the light zone are a mixture of switched and nonswitched cells. Further, the proportion of nonswitched cells, particularly those coexpressing IgD, is higher in GC of mice with impaired CD19 signaling. An exception to this finding is dark zone development in AID-deficient mice, which are unable to switch immunoglobulin class (Muramatsu et al., 2000Muramatsu M. Kinoshita K. Fagarasan S. Yamada S. Shinkai Y. Honjo T. Cell. 2000; 102: 553-563Abstract Full Text Full Text PDF PubMed Scopus (2536) Google Scholar). On the basis of the location of nonswitched cells and 5-bromo-2′-deoxyuridine uptake experiments, the authors postulate that the B cells in the dark zone and light zone may be largely independent pools of proliferating cells. Further, they suggest that affinity maturation occurs in the light zone rather than the dark zone and that proliferation of light zone cells requires continued signals from immune complex, or other ligands on FDC to survive, while those in the dark zone do not. This raises the question about what this hypothetically independent pool of B blasts in the dark zone are doing. Some published data indicate that at least some dark zone blasts move into the FDC network in the light zone. Exchange between these zones is implied from single cell analysis in GC that shows members of the same clone at different stages of mutation in the dark zone and light zone (Küppers et al., 1993Küppers R. Zhao M. Hansmann M.L. Rajewsky K. EMBO J. 1993; 12: 4955-4967PubMed Google Scholar). Previous 5-bromo-2′-deoxyuridine pulse chase experiments in rat GC indicate that B cells that have taken up label in the dark zone during a 3 hr labeling period appear in the light zone between 6 and 10 hr from starting labeling (Liu et al., 1991Liu Y.J. Zhang J. Lane P.J.L. Chan E.Y.-T. MacLennan I.C.M. Eur. J. Immunol. 1991; 21: 2951-2962Crossref PubMed Scopus (591) Google Scholar). Similar findings in mice were reported by Hanna in 1964 (Hanna, 1964Hanna M.G. Lab. Invest. 1964; 13: 95-104PubMed Google Scholar). The period of postlabeling observation of Wang and Carter, 2005Wang Y. Carter R.H. Immunity. 2005; 22 (this issue): 749-761Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar was too brief to pick up this phenomenon. If cells in the dark zone are to be selected it is hard to see how this could happen in situ as antigen and T cells are not obviously present. The data cited above are consistent with these cells passing into the light zone where they are selected. In germinal centers induced in the absence of T cells, dark zones form without the requirement for T cell selection (Vinuesa et al., 2000Vinuesa C.G. Cook M.C. Ball J. Drew M. Sunners Y. Cascalho M. Wabl M. Klaus G.G.B. MacLennan I.C.M. J. Exp. Med. 2000; 191: 485-493Crossref PubMed Scopus (220) Google Scholar). Presumably because antigen available for selection is absent, the B cells in both the light zones and dark zones of these GC undergo involution by mass B cell apoptosis within 5 days of GC induction. If dark zone B cells are not moving to the light zone where they are selected, why should this involution occur in such GC? Hypotheses of the way affinity maturation occurs in GC set out many years ago have been widely accepted, but are not necessarily adequately tested. We must be grateful to Wang and Carter, 2005Wang Y. Carter R.H. Immunity. 2005; 22 (this issue): 749-761Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar for reminding us of the uncertainty that exists about the way B cells are selected during affinity maturation in GC.