Heterogeneity of germinal center B cells: New insights from single-cell studies

Upon antigen exposure, activated B cells in antigen-draining lymphoid organs form microanatomical structures, called germinal centers (GCs), where affinity maturation occurs. Within the GC microenvironment, GC B cells undergo proliferation and B cell receptor (BCR) genes somatic hypermutation in the dark zone (DZ), and affinity-based selection in the light zone (LZ). In the current paradigm of GC dynamics, high-affinity LZ B cells may be selected by cognate T- follicular helper cells to either differentiate into plasma cells or memory B cells, or re-enter the DZ and initiate a new round of proliferation and BCR diversification, before migrating back to the LZ. Given the diversity of cell states and potential cell fates that GC B cells may adopt, the two-state DZ-LZ paradigm has been challenged by studies that explored GC B-cell heterogeneity with a variety of single-cell technologies. Here, we review studies and single-cell technologies which have allowed to refine the working model of GC B-cell cellular and molecular heterogeneity during affinity maturation. This review also covers the use of single-cell quantitative data for mathematical modeling of GC reactions, and the application of single-cell genomics to the study of GC-derived malignancies.     

Germinal center responses to complex antigens

Germinal centers (GCs) are the primary sites of antibody affinity maturation, sites where B-cell antigen-receptor (BCR) genes rapidly acquire mutations and are selected for increasing affinity for antigen. This process of hypermutation and affinity-driven selection results in the clonal expansion of B cells expressing mutated BCRs and acts to hone the antibody repertoire for greater avidity and specificity. Remarkably, whereas the process of affinity maturation has been confirmed in a number of laboratories, models for how affinity maturation in GCs operates are largely from studies of genetically restricted B-cell populations competing for a single hapten epitope. Much less is known about GC responses to complex antigens, which involve both inter- and intraclonal competition for many epitopes. In this review, we (i) compare current methods for analysis of the GC B-cell repertoire, (ii) describe recent studies of GC population dynamics in response to complex antigens, discussing how the observed repertoire changes support or depart from the standard model of clonal selection, and (iii) speculate on the nature and potential importance of the large fraction of GC B cells that do not appear to interact with native antigen.     

Involvement of GANP in B cell activation in T cell-dependent antigen response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.     

CD4<Superscript>+</Superscript> T Cells Downregulate Bcl-2 in Germinal Centers

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2⁺ GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2⁺, in the GC, most T lymphocytes are Bcl-2⁻. In addition, most of the CD4⁺ GC T cells are Bcl-2⁻, while nearly 100% of the CD8⁺ GC T cells are Bcl-2⁺. The Bcl-2 downregulation by both B and CD4⁺ T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.     

T cell interactions with B cells during germinal center formation,a three‐step model

Establishment of effective immunity against invading microbes depends on continuous generation of antibodies that facilitate pathogen clearance. Long‐lived plasma cells with the capacity to produce high affinity antibodies evolve in germinal centers (GCs), where B cells undergo somatic hypermutation and are subjected to affinity‐based selection. Here, we focus on the cellular interactions that take place early in the antibody immune response during GC colonization. Clones bearing B‐cell receptors with different affinities and specificities compete for entry to the GC, at the boundary between the B‐cell and T‐cell zones in lymphoid organs. During this process, B cells compete for interactions with T follicular helper cells, which provide selection signals required for differentiation into GC cells and antibody secreting cells. These cellular engagements are long‐lasting and depend on activation of adhesion molecules that support persistent interactions and promote transmission of signals between the cells. Here, we discuss how interactions between cognate T and B cells are primarily maintained by three types of molecular interactions: homophilic signaling lymphocytic activation molecule (SLAM) interactions, T‐cell receptor: peptide‐loaded major histocompatibility class II (pMHCII), and LFA‐1:ICAMs. These essential components support a three‐step process that controls clonal selection for entry into the antibody affinity maturation response in the GC, and establishment of long‐lasting antibody‐mediated immunity.     

S1PR2 links germinal center confinement and growth regulation

Germinal centers (GCs) are sites of rapid B-cell proliferation and somatic mutation. These ovoid structures develop within the center of follicles and grow to a stereotypic size. The cell migration and interaction dynamics underlying GC B-cell selection events are currently under intense scrutiny. In recent study, we identified a role for a migration inhibitory receptor, S1PR2, in promoting GC B-cell confinement to GCs. S1PR2 also dampens Akt activation and deficiency in S1PR2 or components of its signaling pathway result in a loss of growth control in chronically stimulated mucosal GCs. Herein, we detail present understanding of S1PR2 and S1P biology as it pertains to GC B cells and place this information in the context of a current model of GC function.     

Impaired clearance of apoptotic cells in germinal centers: implications for loss of B cell tolerance and induction of autoimmunity

Germinal centers (GCs) comprise lymphoid microenvironments where antigen-stimulated B cells undergo rapid proliferation and somatic hypermutation (SHM), resulting in the generation of B cells with high affinity for antigen. However, this process also generates B cell clones with low antigen affinity and with the potential for autoreactivity. It has been suggested that GC B cells with low antigen affinity and autoreactivity are eliminated via apoptosis and are rapidly cleared by tingible body macrophages (TBMφs). Inefficient clearance of apoptotic cells (ACs) results in autoimmunity that is thought to be mediated by various intracellular molecules possessing danger-associated molecular patterns (DAMPs), including nuclear self-Ags. DAMPs can be released from ACs undergoing "secondary necrosis" due to a disruption in AC clearance within GCs. This review discusses the role and mechanisms associated with impaired clearance of ACs in GCs in loss of B cell tolerance leading to autoantibody production and the development of autoimmunity.     

Role of BCR affinity in T cell dependent antibody responses in vivo

Antibody affinity for antigen is believed to govern B lymphocyte selection during T-dependent immune responses. To examine antibody affinity in T cell dependent immune responses, we compared mice that carry targeted V(H)B1-8 antibody genes with high or low antigen-binding affinity. We found that high- and low-affinity B cells had the same intrinsic capacity to respond to antigen, but in experiments where limiting numbers of high- and low-affinity B cells were mixed in wild-type recipient mice, only the high-affinity B cells accumulated in germinal centers (GCs). In GCs, high-affinity B cells accumulated fewer V(H) somatic mutations than low affinity B cells. This effect was due to selections as the frequency of mutation in noncoding immunoglobulin gene DNA is the same in high- and low- affinity B cells. Thus, B cells recruited to the GC appeared to undergo a fixed mutation program, regardless of initial B cell receptor affinity. We conclude that in addition to the selection that occurs in GCs, stringent selection for high-affinity clones is also imposed in the early stages of the T cell dependent immune response in vivo.     

Absence of interleukin-4 enhances germinal center reaction in secondary immune response

The germinal center (GC) is a compartment for B cell differentiation and proliferation. Interleukin (IL)-4 has been considered essential for GC functioning. To define the role of IL-4 in GC reaction, immunohistology of draining lymph nodes (LNs) of IL-4 gene-targeted (IL-4(-/-)) mice was performed during secondary immune response. IL-4(-/-) mice were immunized with ovalbumin emulsified in Freund' complete adjuvant. Final antigen challenge was done 4 weeks later. IL-4(-/-) mice had a higher production of IgG2a and IgG2b and a lower production of IgG1 than those in wild-type (WT) mice. In comparison with WT mice, LNs of IL-4(-/-) mice on days 4 and 7 after final antigen challenge were larger and contained a markedly greater number of GCs, which showed marked size variations with a large number of small GCs and a small number of markedly large GCs. By day 14, the number of GCs decreased to the same level as that in WT mice. However, the LN size in IL-4(-/-) mice was still larger than that in WT mice due to the presence of markedly large GCs. Although well-developed complement receptor(+) follicular dendritic cell (FDC) networks were present in GCs of IL-4(-/-) mice, no FDCs of mature phenotype (CD23(+)) were observed in many of the small GCs. In conclusion, the absence of IL-4 enhanced GC reaction and specific antibody response of Th1-type. IL-4 may play an important role in inducing the appropriate magnitude of humoral immune response.     

Origin and characteristics of glycogen cells in the developing murine placenta.

The junctional zone (Jz) of the mouse placenta consists of two main trophoblast populations, spongiotrophoblasts and glycogen cells (GCs), but the development and function of both cell types are unknown. We conducted a quantitative analysis of GC size, number, and invasion of cells into the decidua across gestation. Furthermore, we identified markers of GC function to investigate their possible roles in the placenta. While the spongiotrophoblast cell volume doubles, and cell number increases steadily from E12.5 to E16.5, there is a remarkable 80-fold increase in GC numbers. This finding is followed by a notable decrease by E18.5. Surprisingly, the accumulation of GCs in the decidua did not fully account for the decrease in GC number in the Jz, suggesting loss of GCs from the placenta. Glucagons were detected on GCs, suggesting a steady glucose release throughout gestation. Connexin31 staining was shown to be specific for GCs. GC migration and invasion may be facilitated by temporally regulated expression of matrix metalloproteinase 9 and the imprinted gene product, Decorin. Expression of the clearance receptor for type II insulin-like growth factor (IGF-II), IGF2R, in a short developmental window before E16.5 may be associated with regulating the growth effects of IGF-II from glycogen cells and/or labyrinthine trophoblast on the expansion of the Jz. Thus stereology and immunohistochemistry have provided useful insights into Jz development and function of the glycogen cells.     

From SAP-less T cells to helpless B cells and back: dynamic T-B cell interactions underlie germinal center development and function

Operation of the immune system critically depends on intercellular communication among multiple cell types, frequently in the form of physical cell-cell interactions. Germinal centers (GCs) are highly organized tissue microdomains in which high affinity, class-switched, antibody-producing cells and humoral immune memory are generated. Critical underlying cell-cell interaction events include at the minimum binary interactions between CD4(+) T-helper cells and antigen-presenting dendritic cells (DCs), which ensure proper T-cell activation and acquisition of effecter potentials, and those between T-helper cells and antigen-activated B cells whereby the latter cells receive helper signals (e.g. CD40L) important for their proliferation, survival, and differentiation. How these critical cellular interaction events are molecularly regulated and dynamically orchestrated to support GC formation and function is still a study in progress. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) has recently been defined as a pivotal molecule that controls cognate T-B interactions and GC formation. Detailed analysis of interaction and migration dynamics of SAP-deficient T cells has raised the interesting possibility that T cell:antigen-presenting cell interactions underlying GC development and function are regulated in a cell type- and spatiotemporal stage-specific manner. This has important implications for our understanding of synapse formation, helper signal delivery to B cells, follicular helper T-cell differentiation, and quality control of the GC reaction in general. A model of selective T-B interactions involving bi-directional feedback and feed-forward logic is proposed to underlie GC development and function.     

Single-cell PCR analysis of T helper cells in human lymph node germinal centers

The T helper cell population of human lymph node germinal centers (GCs) was analyzed for clonality and signs of antigen selection. Frozen sections of lymph node biopsies taken from three different individuals were used to micromanipulate single T cells from one particular GC for each of the specimens. T cell receptor (TCR) beta gene rearrangements were amplified from these single cells and directly sequenced. Although only unique rearrangements were amplified from T cells of GC2 and GC3, 11 of 28 potentially functional rearrangements amplified from GC1 originated from four different clones. In all three GCs, TCR gene rearrangements neither showed obvious biases in gene segment usage nor similarities in complementarity determining region 3 amino acid sequence. Thus, it appears that T lymphocytes in human GCs usually represent a diverse population of cells. Sequence analysis of V region genes did not provide evidence that in the human the process of somatic hypermutation acts on the TCRbeta loci. For one of the GCs (GC3), immunoglobulin heavy chain (IgH) gene rearrangements were amplified and sequenced from single micromanipulated GC B cells. The detection of clonal expansions accounting for more than half of the sampled B cells in addition to ongoing somatic hypermutation of Ig V region genes suggested that GC3 was a fully developed GC.     

Plasma cell differentiation during the germinal center reaction

Germinal centers (GCs) are formed in secondary lymphoid tissues upon immunization with T‐dependent antigens. In GCs, somatic hypermutation generates B cells with increased antibody affinity and these high‐affinity B cells preferentially differentiate into plasma cells, which home to bone marrow and confer long‐lived humoral immunity. Recent studies have shed new light on the cellular and molecular basis for initiating the transition from a GC B cell to a plasma cell. Here, we review recent progress in our understanding of how plasma cell generation during the GC reaction is regulated for inducing effective long‐term protective immunity and for preventing harmful autoimmunity.     

Molecular mechanisms insulating proliferation from genotoxic stress in B lymphocytes

In mammals, B cells strictly segregate proliferation from somatic mutation as they develop within the bone marrow and then mature through germinal centers (GCs) in the periphery. Failure to do so risks autoimmunity and neoplastic transformation. Recent work has described how B cell progenitors transition between proliferation and mutation via cytokine signaling pathways, epigenetic chromatin regulation, and remodeling of 3D chromatin conformation. We propose a three-zone model of the GC that describes how proliferation and mutation are regulated. Using this model, we consider how recent mechanistic discoveries in B cell progenitors inform models of GC B cell function and reveal fundamental mechanisms underpinning humoral immunity, autoimmunity, and lymphomagenesis.     

Human germinal center T cells are unique Th cells with high propensity for apoptosis induction

Collaborative interactions between T(h) cells and B cells are necessary for the production of antibody responses to most protein antigens and for the generation of memory B cells in germinal centers (GCs). Although it is well established that T(h) cells are pivotal for the GC reaction, the mechanisms that control the homeostasis of T(h) cells during the GC response remain largely unknown. Here we show that, unlike other effector T cells, a significant number of CD4(+)CD45RO(+)CD57(+) T cells, which are the major T(h) cells residing in the GCs, are undergoing apoptosis in vivo. CD4(+)CD45RO(+)CD57(+) GC T cells exhibit similar sensitivities to apoptotic signals and to caspase inhibitors as immature thymocytes. Moreover, CD4(+)CD45RO(+)CD57(+) GC T cells express a unique profile of genes that control apoptosis and cell cycle, providing possible molecular mechanisms for the high rates of apoptotic death of these T(h) cells in the GCs.     

Comparison of IgD+ and IgD- thoracic duct B lymphocytes as germinal center precursor cells in the rat.

Genetically marked thoracic duct B cell subpopulations rich in either IgD+ or IgD- B cells were transferred to non-irradiated, congenic rats in order to compare the capacities of IgD+ versus IgD- B cells to form germinal centers (GCs). This comparison was made quantitatively based on flow cytometric analyses of lymph node cells prepared from chimeric rats 7 days after s.c. immunization. Donor-origin and host-origin B cells were distinguished using anti-Igk antibodies, and GC B cells were distinguished from other B cells in suspension by their lack of labeling with the mAb HIS22. IgK+ HIS22- lymph node cells corresponded well to GC B cells: they contained many large cells, were IgM+ but mostly IgD-, expressed relatively lower levels of IgM than HIS22+ B cells, and increased in number and frequency in response to antigen. Results from flow cytometric analyses, corroborated by immunofluorescence histochemical studies, showed that cell-for-cell, IgD- B cells from GCs much more efficiently than IgD+ cells. B cell populations enriched for IgD- cells became relatively more distributed to GCs than to other lymph node B cell areas and gave rise to many more GC B cells of donor origin per transferred B cell than whole, unseparated thoracic duct B cells (for which greater than 97% were IgD+). IgD- B cells from rats primed deliberately with antigen also became relatively more distributed to GCs and gave rise to more GC B cells of donor origin than either IgD+ B cells from primed donors or IgD- B cells from unprimed donors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinogen is localized on dark zone follicular dendritic cells in vivo and enhances the proliferation and survival of a centroblastic cell line in vitro

Follicular dendritic cells (FDC) in the germinal centers (GC) of secondary lymphoid organs increase the survival and proliferation of antigen-stimulated B cells and are pivotal for the affinity maturation of an antibody response and for maintenance of B cell immunological memory. The dark zone (DZ) and the light zone (LZ) constitute distinct areas of the GC containing different subtypes of FDC as identified by their morphology and phenotype. Until now, most available FDC-specific reagents identify LZ FDC, and there are no reagents recognizing DZ FDC specifically. Here, we report a new mAb, D46, which stains FDC specifically in the DZ of bovine and ovine GC within the secondary follicles. We identify its ligand as bovine fibrinogen, and using commercially available anti-human fibrinogen antibodies, show that this inflammatory protein is also present on DZ FDC of human GC within palatine tonsils. In vitro, the addition of exogenous fibrinogen stimulates the proliferation and survival of BCR-stimulated L3055 cells, which constitute a clonal population of centroblastic cells and retain important features of normal GC B cells. Together, our results suggest that fibrinogen localized on DZ FDC could support the extensive proliferation and survival of GC B cells within the DZ in vivo.     

Cellular origin of human B-cell neoplasms and Hodgkin's disease based on analysis of somatic hypermutations in the immunoglobulin variable region genes

In response to antigen stimulation, B cells undergo a germinal center(GC) reaction such as somatic hypermutations of the immunoglobulin variable region genes, which results in the production and selection of antigen-specific antibodies with increased affinity. Therefore, somatic hypermutations are considered to be a hallmark of GC B cells and their descendants. Pre-GC B cells(precursor B cells, immature B cells, naive B cells and CD5+ B cells) carry no somatic hypermutations, whereas GC B cells and post-GC B cells(memory B cells and plasma cells) express somatic hypermutations. This phenomenon is useful in identifying the cellular origin of various B-cell neoplasms. Precursor B-lymphoblastic leukemia/lymphoma, mantle cell lymphoma, and most B-CLL originate from pre-GC B cells, and follicular lymphoma, Burkitt's lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma and myeloma from GC B cells or post-GC B cells. Nodular lymphocyte-predominant Hodgkin's disease and most classical types of Hodgkin's disease are derived from GC B cells. Most human-B cell neoplasms including Hodgkin's disease are derived from GC B cells or their descendants. Molecular processes that modify the DNA of GC B cells, such as somatic hypermutation, class switching and receptor editing occur in the environment of the GCs, and increase the risk of malignant transformation.