steven.kleinstein@yale.edu.

Summary We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1 and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by one day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 were no longer expressed on follicular Bcl6hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1hi ICOShi GL7hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF) and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS resident B cells encounter antigen and experience affinity maturation. In this study, paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clonal families were more often found in the draining CLNs. More mature clonal family members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or impact the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.

Analyses of somatic hypermutation (SHM) patterns in B cell immunoglobulin (Ig) sequences contribute to our basic understanding of adaptive immunity, and have broad applications not only for understanding the immune response to pathogens, but also to determining the role of SHM in autoimmunity and B cell cancers. Although stochastic, SHM displays intrinsic biases that can confound statistical analysis, especially when combined with the particular codon usage and base composition in Ig sequences. Analysis of B cell clonal expansion, diversification, and selection processes thus critically depends on an accurate background model for SHM micro-sequence targeting (i.e., hot/cold-spots) and nucleotide substitution. Existing models are based on small numbers of sequences/mutations, in part because they depend on data from non-coding regions or non-functional sequences to remove the confounding influences of selection. Here, we combine high-throughput Ig sequencing with new computational analysis methods to produce improved models of SHM targeting and substitution that are based only on synonymous mutations, and are thus independent of selection. The resulting “S5F” models are based on 806,860 Synonymous mutations in 5-mer motifs from 1,145,182 Functional sequences and account for dependencies on the adjacent four nucleotides (two bases upstream and downstream of the mutation). The estimated profiles can explain almost half of the variance in observed mutation patterns, and clearly show that both mutation targeting and substitution are significantly influenced by neighboring bases. While mutability and substitution profiles were highly conserved across individuals, the variability across motifs was found to be much larger than previously estimated. The model and method source code are made available at