• Architecture of the primary immune response: Phenotypic and molecular genetic changes in antigen-driven B cell populations

      Jacob, Joshy; Kelsoe, Garnett, 1949- (1992)
      Much of our understanding of B cells and the immune response is based primarily upon the study of dissociated lymphocytes. If the observations made in vitro are to be related to events occurring in vivo, it is crucial to study antigen-activated cells in their architectural context. To this end, I have investigated the clonally restricted immune response of {dollar}Igh\sp{lcub}b{rcub}{dollar} mice to immunogenic forms of the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) as it occurs in vivo, by analyzing serial spleen sections. Following immunization, two populations of antigen-activated B cells are seen in the spleen; foci and germinal centers (GC). Foci and GC are discrete areas of intense B cell proliferation and are oligoclonal; each founded on average by 1-3 precursor cells. Using reagents specific for the hallmarks of this response and DNA sequence analysis of individual NP-specific foci and GC B cells, I have attempted to detail the architecture, clonal dynamics and somatic diversification of an immune response. Even though foci and GC arise as a common consequence of immunization, these two B cell populations are distinct; they differ in their anatomical location, growth kinetics and phenotype. Foci appear in the T cell-rich periarteriolar lymphoid sheaths in the white pulp, prior to the appearance of GC in the follicles. B cells in foci do not bind PNA; while GC B cells avidly bind this lectin. While isotype switching (IgM {dollar}\to{dollar} IgG) occurs in both foci and GC, somatic mutation is restricted to the GC microenvironment. Somatic mutation is initiated in GC B cells at days 7-8 postimmunization and mutations are introduced into the IgV genes at random in a stepwise manner. Thus foci represent populations of antigen-driven selection among unmutated B cells while GC are populations of selection among mutated sister B cells which presumably give rise to memory B cells. Interestingly, B cells in adjacent foci and GC have a common clonal origin based upon comparison of the third complementarity determining region (CDR) of the H chain suggesting that GC are most likely founded by antigen-activated B cells in neighboring focus. Thus antigen-activated B cells appear to have the potential to follow different differentiation pathways and microenvironmental influences determine the fate of B cells in vivo.
    • Clonal rivalry: The role of affinity and competition in B cell activation and differentiation during humoral immune responses

      Dal Porto, Joseph Matthew; Kelsoe, Garnett, 1949- (1998)
      The vertebrate humoral immune system is extraordinary in both its capacity to mount a response to virtually any foreign antigen and in its ability to produce antibody of remarkable specificity and affinity for that antigen. However, primary exposure to antigen initially leads to the production of antibody with significant heterogeneity in affinity, including those with astonishingly low affinity (approx 10 to the 4th-10 to the 5th Ml). As these are affinity values within the realm of non-specific protein-protein interactions, many investigators have doubted the authenticity of such low affinity antibody and suggested that their presence is artifactual, i.e., due to non-specific, or bystander, activation. Are these extraordinarily low affinity antibodies the result of antigen-specific activation? Further, while these low affinity antibodies are detectable early after challenge with antigen, they are noticeably absent from the later stages of the response. Are there affinity requisites for levels of activation and differentiation of B cells in humoral responses, ones that low affinity cells are intrinsically incapable of achieving? Or, alternatively, does clonal rivalry, competition between B cells of varying affinity for declining amounts of antigen, reduce the likelihood that low affinity B cells will survive in the presence of higher affinity competitors? I have investigated the role of B cell affinity at the onset and during the propagation of humoral immune responses. I examined the affinity of early antigen-activated B cells in mice after immunization by recovery and re-expression of representative VDJ rearrangements as immunoglobulin molecules. Interestingly, the average affinity of these initial B cells was determined to be extremely low (Ka approx 4 x 10 to the 5th M1), a value comparable to the binding interaction of T cell receptors for MHC and peptide. These results suggest that antigen normally activates an unexpectedly wide spectrum of B cells, including those with exceptionally low affinities. To test this model, two lines of immunoglobulin heavy chain (IgH) transgenic mice were created which express very low affinity immunoglobulin receptors. Even with extraordinarily low affinity BCR, immunization of transgenic mice led to specific humoral responses with kinetics similar to those observed in wild-type controls, including an increase in antigen-specific serum immunoglobulin, the production of splenic antibody-forming cells (AFC) and germinal centers (GCs). Finally, I investigated whether affinity controls entry into the B-cell memory pool by assessing the memory response in low affinity transgenic mice. My results demonstrate that selection into the memory compartment can be extremely lenient in regard to affinity for antigen and is most likely controlled by competition for survival taking place in GC of the primary response. These results suggest that clonal selection may in fact represent clonal rivalry for occupancy of immunological niches during an immune response and affinity maturation is the result of darwinian evolution for survival of the fittest.