Mary M. Tomayko

PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells

Memory B and plasma cells (PCs) are generated in the germinal center (GC). Because follicular helper T cells (TFH cells) have high expression of the immunoinhibitory receptor PD-1, we investigated the role of PD-1 signaling in the humoral response. We found that the PD-1 ligands PD-L1 and PD-L2 were upregulated on GC B cells. Mice deficient in PD-L2 (Pdcd1lg2−/−), PD-L1 and PD-L2 (Cd274−/−Pdcd1lg2−/−) or PD-1 (Pdcd1−/−) had fewer long-lived PCs. The mechanism involved more GC cell death and less TFH cell cytokine production in the absence of PD-1; the effect was selective, as remaining PCs had greater affinity for antigen. PD-1 expression on T cells and PD-L2 expression on B cells controlled TFH cell and PC numbers. Thus, PD-1 regulates selection and survival in the GC, affecting the quantity and quality of long-lived PCs.

New markers for murine memory B cells that define mutated and unmutated subsets

The study of murine memory B cells has been limited by small cell numbers and the lack of a definitive marker. We have addressed some of these difficulties with hapten-specific transgenic (Tg) mouse models that yield relatively large numbers of antigen-specific memory B cells upon immunization. Using these models, along with a 5-bromo-2′-deoxyuridine (BrdU) pulse-label strategy, we compared memory cells to their naive precursors in a comprehensive flow cytometric survey, thus revealing several new murine memory B cell markers. Most interestingly, memory cells were phenotypically heterogeneous. Particularly surprising was the finding of an unmutated memory B cell subset identified by the expression of CD80 and CD35. We confirmed these findings in an analogous V region knock-in mouse and/or in non-Tg mice. There also was anatomic heterogeneity, with BrdU+ memory cells residing not just in the marginal zone, as had been thought, but also in splenic follicles. These studies impact the current understanding of murine memory B cells by identifying new phenotypes and by challenging assumptions about the location and V region mutation status of memory cells. The apparent heterogeneity in the memory compartment implies either different origins and/or different functions, which we discuss.

BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

Cutting Edge: Hierarchy of Maturity of Murine Memory B Cell Subsets

The paucity of murine memory B cell markers has been a significant impediment to the study of memory. The most commonly used marker is IgG, which is neither sensitive nor specific, because activated nonmemory cells can be IgG+, and memory cells can be IgM+. In this article, we show that, together, PD-L2 (CD273), CD80, and CD73 define at least five phenotypic subsets of murine memory B cells. These subsets are generated from naive cells bearing a single BCR in response to a single T-dependent Ag. This diversity is independent of class switch, because IgG1- and IgM-bearing memory cells are found within each compartment. Memory subsets defined by PD-L2, CD80, and CD73 are biologically distinct from one another, because they differ in ontogeny and selection. Together, these distinctions suggest that there is a spectrum of memory B cells and progressive acquisition from more naive-like to more memory-like properties.

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype

Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80+PD-L2+ MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80−PD-L2− MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.

Systematic Comparison of Gene Expression between Murine Memory and Naive B Cells Demonstrates That Memory B Cells Have Unique Signaling Capabilities

Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naive precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of Ag-specific memory and naive cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. We identified genes with differential expression and confirmed the differential expression of many of these by quantitative RT-PCR and of some of these at the protein level. Our initial analysis revealed differential expression patterns of genes that regulate signaling. Memory B cells have increased expression of genes important in regulating adenosine signaling and in modulating cAMP responses. Furthermore, memory B cells up-regulate receptors that are essential for embryonic stem cell self-renewal. We further demonstrate that one of these, leukemia inhibitory factor receptor, can initiate functional signaling in memory B cells whereas it does not in naive B cells. Thus, memory and naive B cells are intrinsically wired to signal differently from one another and express a functional signaling pathway that is known to maintain stem cells in other lineages.

Intrinsic properties of human and murine memory B cells.

Summary:  The central question of how the immune system responds in a qualitatively and quantitatively better way upon re‐exposure to a pathogen is largely unanswered. Both the increased frequency of antigen‐specific memory cells and the intrinsic properties that memory cells acquire after antigen experience could contribute to the faster and more robust responses seen after repeated exposure to antigen. In the case of the memory B‐cell response, it has been difficult to discern the individual contributions of these two effects. However, because of recent advances in identifying memory B cells, there is an increasing understanding of the intrinsic properties of these cells. The current insights into the unique properties of memory B cells and the progress that has been made in understanding how these affect secondary responses in both the human and the mouse systems are discussed. In addition, we compare the various advantages and disadvantages inherent in each of these systems, in terms of studying the intrinsic properties of memory B cells, and introduce the details of the system that we have developed using conventional heavy chain transgenic (Tgic) mice, which addresses some of the drawbacks of traditional memory models.

Multiple Transcription Factor Binding Sites Predict AID Targeting in Non-Ig Genes

Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ∼25% of expressed genes in germinal center B cells. Observations in Ung−/− Msh2−/− mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung−/− Msh2−/− mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-β binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID.

Development of bullous pemphigoid during nivolumab therapy

Bregs: B regulatory cells BP: bullous pemphigoid CTLA-4: cytotoxic T-lymphocyteeassociated protein 4 IRAE: immune-related adverse events PD-1: programmed cell death protein 1 PD-L1: programmed death ligand

CD73 Expression Is Dynamically Regulated in the Germinal Center and Bone Marrow Plasma Cells Are Diminished in Its Absence

CD73 catalyzes the conversion of extracellular nucleosides to adenosine, modulating inflammatory and T cell responses. Elevated expression of CD73 marks subpopulations of murine memory B cells (MBC), but its role in memory development or function is unknown. Here, we demonstrate that CD73 is progressively upregulated on germinal center (GC) B cells following immunization, is expressed at even higher levels among T follicular helper cells, but is absent among plasma cells (PC) and plasmablasts (PB). We analyzed the T-dependent B cell response in CD73 knockout mice (CD73KO). During the early response, CD73KO and wild type (WT) mice formed GCs, MBCs and splenic PBs and PCs similarly, and MBCs functioned similarly in the early secondary response. Late in the primary response, however, bone marrow (BM) PCs were markedly decreased in CD73KO animals. Tracking this phenotype, we found that CD73 expression was required on BM-derived cells for optimal BM PC responses. However, deletion of CD73 from either B or T lymphocytes alone did not recapitulate the phenotype. This suggests that CD73 expression is sufficient on either cell type, consistent with its function as an ectoenzyme. Together, these findings suggest that CD73-dependent adenosine signaling is prominent in the mature GC and required for establishment of the long-lived PC compartment, thus identifying a novel role for CD73 in humoral immunity.