A Multiscale Spatial Modeling Framework for the Germinal Center Response.

Follicular dendritic cells (FDC) form the framework of the germinal centre (GC). They act as accessory cells, by providing a favourable microenvironment for the germinal centre reaction.1-2 Secondary GCs consist of a basal dark zone, mainly composed of immature blast cells and showing extensive mitotic activity, and an apical light zone containing a heterogeneous population of lymphoid cells. FDC are present both in the dark zone and in the light zone of the GC. Only few properties of the FDC present in these zones are known. The FDC density framework shows only minor differences between the dark and the light zone. Cytochemical localization of immune-complexes and the ectoenzyme 5’-nucleotidase indicate differences in the expression of these markers in FDC in these zones.3 These staining patterns suggest differences in the functional stage of FDC. To date, the ultrastructural morphology of FDC in relation to these functionally different GC compartments is not well established. In human GCs the different types of lymphoid and non-lymphoid cells can be distinguished on the basis of their ultrastructure.4 We performed a qualitative and quantitative ultrastructural study of the FDC population in the dark and the light zones of GCs. This approach was aimed to define the light and the dark zones of GCs, and to study - on a structure-function basis -the contribution of FDC to these distinct microenvironments.KeywordsGerminal CentreRough Endoplasmic ReticulumFollicular Dendritic CellDense DepositDark Zone

The germinal center (GC) is divided into a dark zone (DZ) and a light zone (LZ). GC B cells must cycle between these zones to achieve efficient Ab affinity maturation. Follicular dendritic cells (FDCs) are well characterized for their role in supporting B cell Ag encounter in primary follicles and in the GC LZ. However, the properties of stromal cells supporting B cells in the DZ are relatively unexplored. Recent work identified a novel stromal population of Cxcl12-expressing reticular cells (CRCs) in murine GC DZs. In this article, we report that CRCs have diverse morphologies, appearing in open and closed networks, with variable distribution in lymphoid tissue GCs. CRCs are also present in splenic and peripheral lymph node primary follicles. Real-time two-photon microscopy of Peyer's patch GCs demonstrates B cells moving in close association with CRC processes. CRCs are gp38(+) with low to undetectable expression of FDC markers, but CRC-like cells in the DZ are lineage marked, along with FDCs and fibroblastic reticular cells, by CD21-Cre- and Ccl19-Cre-directed fluorescent reporters. In contrast to FDCs, CRCs do not demonstrate dependence on lymphotoxin or TNF for chemokine expression or network morphology. CRC distribution in the DZ does require CXCR4 signaling, which is necessary for GC B cells to access the DZ and likely to interact with CRC processes. Our findings establish CRCs as a major stromal cell type in the GC DZ and suggest that CRCs support critical activities of GC B cells in the DZ niche through Cxcl12 expression and direct cell-cell interactions.

The structure of germinal centres (GCs) in human secondary lymphatic organs has not been thoroughly investigated until now. We stained follicular dendritic cells (FDCs) in serial sections of human hyperplastic tonsils and spleens to compare the morphology of GCs in fulminant immune reactions and quiescence. Detection of CD35, CD21, CD23 and the target of mAb CNA.42 confirmed that full-blown human tonsil GCs may consist of four regions, the dark zone, the basal and apical light zone and the outer zone. The outer zone was, however, not a constant feature of tonsillar GCs and existed only in a minority of follicles in most specimens. Thus, between 3 and 60% of tonsil GCs with a CD23(+) apical light zone exhibited an outer zone in individual specimens. FDCs in tonsil GCs appeared to be extremely sensitive to mechanical stress during surgery. In contrast to tonsils, seven of the eight adult spleens did not exhibit asymmetric polarized GCs, but only symmetric GCs without dark and light zones or follicles with few GC B cells. Some specimens apparently only contained primary follicles after conventional staining, but on closer inspection a homogenous hyaline extracellular material deposited among the FDCs indicated that a GC had been present. Our study demonstrates that the structure of GCs varies in different human secondary lymphatic organs most likely depending on the local antigenic challenge.

Chapter 23 - The Regulation of IgA Production

Follicular dendritic cells help advance B-Cells in becoming memory B-Cells or antibody-producing plasma cells in the light zone, or undergo additional affinity maturation in the dark zone. Follicular dendritic cell sarcoma (FDCS) is an extremely rare soft tissue malignancy derived from follicular dendritic cells. Autoimmune disease increases the risks for the development of hematological malignancies. To the best of our knowledge, there are few cases of FDCS development in the setting of underlying Sjogren's syndrome (SS). Therefore, in this report, we present a novel case of FDCSassociated with new-onset SS. In SS, the follicular dendritic cells are organized within germinal centers within the glands it infiltrates and is involved in B-Cell development. Because FDCS is derived from follicular dendritic cells, our report postulates that the unregulated follicular dendritic cell proliferation that may occur in SS could increase the risk for FDCS. Due to this possible connection observed in our patient, we highlight FDCS as a differential diagnosis when considering soft tissue cancers. We urge additional research to outline and explore the possible pathologic link between SS and FDCS.

The germinal center reaction is the process by which low-affinity B cells evolve into potent, immunoglobulin-secreting plasma and memory B cells. Since the recycling hypothesis was created, experimental studies have both tracked movement of a small population of B cells from the light zone into the dark zone, supporting the recycling model, and parallel to the light zone–dark zone interface, indicating a one-way trajectory. We present a novel, sequence-based ab initio model of protein stability and protein interactions. Our model contains a dark zone region of clonal expansion and somatic hypermutation and a light zone site of antigenic selection. We show not only that a one-shot model is sufficient to achieve biologically-realistic rates of affinity growth, population dynamics, and silent:non-silent mutation ratios in the complementary determining region and framework region of antibodies, but also that a stochastic recycling program with or without realistic constraints on the structural stabilities of GC antibodies cannot produce biologically-observed affinity growth, population dynamics or silent:non-silent mutation profiles. The effect of recycling erases affinity gains made by potent antibodies cycling back from the light zone and causes B cells to pool in the dark zone under high replication rates.

The expression of adhesion molecules on human tonsillar follicular dendritic cells (FDCs) in vivo on cryostat sections and in vitro on isolated FDCs on cytospin preparations was studied. Isolation of FDCs was performed using a magnetic cell sorter (MACS). Immunochemically, FDCs were positive for Mac-1 (CD11b), sialyl-Lex (CD15s), CD22, integrin β1 (CD29), CD40, VLA-α3 (CD49c), VLA-α5 (CD49e), VLA-α6 (CD49f), ICAM-3 (CD50), ICAM-1 (CD54), B7 (CD80), and VCAM-1 (CD106). These adhesion molecules, except ICAM-3 (CD50) which was weakly positive only in the light zone (LZ), were positive with a lacy pattern in all zones of secondary lymphoid follicle, especially strong in the LZ. ICAM-3 (CD50) was positive on more than half of isolated FDCs, but other molecules were positive on almost all isolated FDCs. Concerning the ligands on B cells for these adhesion molecules, the Mac-1 (CD11b)-ICAM-1 (CD54), the ICAM-3 (CD50)-LFA-1 (CD11a/18), and the VCAM-1 (CD106)-VLA-4 (CD29/49d) interactions in the LZ, the sialy-Lex (CD15s)-L-selectin (CD62L) and the VCAM-1 (CD106)-VLA-4 (CD29/49d) interactions in the mantle zone, and the ICAM-1 (CD54)-LFA-1 (CD11a/18) interaction in the entire lymphoid follicle may participate in FDC-B cell adhesion. Furthermore, above mentioned immunohistochemical evidence that FDCs were positive for fibronectin-receptor (CD29/CD49e) and laminin-receptor (CD29/CD49f), was confirmed with a frozen-section binding assay, using isolated FDCs and cryostat sections of tonsils. The numbers of FDCs binding to germinal centers (GCs) were reduced remarkably by pretreatment with monoclonal antibodies for CD29 (32.8±4.1% of the control), CD49e (29.7±2.5%), and CD49f (18.8±0.8%). These data clearly indicated that FDCs bind to the reticulin or laminin fiber in GCs via either receptor.

Lymphoid tissues are composed of B-cell dependent area (lymphoid follicle), and T-cell dependent paracortical and interfollicular areas. In lymphoid follicles, B lymphocytes and follicular dendritic cells (FDCs) play a central role of humoral immunity. B lymphocytes mature to memory B cells or plasmablasts through the proliferation in the dark zone (DZ), the selection in the basal light zone (BLZ), and the differentiation in the apical light zone (ALZ). In these processes, the importance of many cytokines has been pointed.There is, however, little information in the interature about the cytokine receptors in lymphoid follicles. The aim of our study was to investigate the localization of cytokines and theirreceptorsinhuman lymphoid tissues using immunohistochemical and immunocytochemical methods. FDCs were positive for transforming growth factor beta receptor type II (TGF-beta R II) in ALZ, interleukin 1 receptor type II (IL-1 R II, CD121a), interleukin 2 receptor beta (IL-2 R-beta, CD122), interleukin 4 receptor (IL-4R, CD124), interleukin 6 receptor (IL-6R, CD126), granulocyte macrophage-colony stimulating factor receptor (GM-CSF R, CD116w) in the BLZ and ALZ. Many cytokine inverstigated in this study were negative on FDCs except the TGF-beta, but positive on many T cells in lymphoid follicles.Furthermore, we investigated the relationship between IgA positive plasma cells and the expression of TGF-beta and its receptor in lymphoid follicles. The data indicated that lymphoid follicles containing more IgA-positive plasma cells tended to be, simultaneously, more frequently labeled by TGF-beta and its receptor.These data indicated that cytokines produced by T-cells not only may directly affect on B-cells, but also indirectly via FDCs expressing receptors for some cytokines. Simultaneous evaluation of both cytokines and their receptors may provide a powerful strategy of clarifying the cytokine network and transduction in lymphoid follicles.

Class Switch Recombination and Somatic Hypermutation in Early Mouse B Cells Are Mediated by B Cell and Toll-like Receptors

Germinal centers (GCs) are sites for antibody diversification, somatic hypermutation (SHM) and antigen specific B cell selection. GCs are anatomically divided into two distinct areas known as dark zone (DZ) and light zone (LZ) where SHM and clonal selection occurs, respectively. Although previous studies have suggested a B cell-intrinsic role of STAT3 in GC maintenance and antibody responses, how STAT3 signaling in B cells may maintain GCs for B cell selection is not clear. Using immunization and infection models, we show that STAT3 in B cells regulates the GC DZ, resulting in decreased high affinity antibody production. STAT3 deficient DZ B cells demonstrate a decreased cell proliferation and increased apoptotic activity. Further, STAT3 deletion in B cells led to defects in plasma cell formation and B cell memory response. Using GC B cell specific STAT3 conditional knockout mice and GC B cell culture system, we show that STAT3 deficient GC B cells display low amounts of FOXO1, a critical transcription factor required for GC DZ program. Transcriptomic analysis of GC B cells from STAT3 deficient and sufficient mice reveals STAT3-mediated gene regulation of multiple cellular pathways known to be involved in GC DZ maintenance. In conclusion, STAT3 signaling in B cells controls the GC DZ, which, in turn, helps maintain GCs for high-affinity antibody production. Our findings identify STAT3 as a novel target for enhancing humoral immunity as well as for the treatment of diseases involving abnormal GC activity.

TuJ1 (class III β-tubulin) expression suggests dynamic redistribution of follicular dendritic cells in lymphoid tissue

The germinal center (GC) is composed of distinct regions with proliferation and affinity maturation of GC B cells (GCB) occurring in the dark zone (DZ) and antigen-mediated selection and commitment of the long-lived plasma cell (LL-PCs) precursors occurring in the light zone (LZ). Despite the key role for GCs in establishing enduring humoral immunity, we know little about the dynamics of the GCB cell response. We showed that the transcription factor T-bet is expressed by GCB cells and that B cell intrinsic T-bet is required for the LL-PC response following influenza (flu) infection. To test whether the loss of LL-PCs in these mice was due to a role for T-bet in regulating the GCB response, we infected 50:50 Tbx21 −/−: pepboy chimeric mice with flu and enumerated GCB cells. We found that Tbx21 −/− B cells were overrepresented in the GC relative to the pepboy cells and that Tbx21 −/− GCB cells were enriched in the LZ. Partitioning of GCB cells in the LZ/DZ is regulated by chemokines and we showed that T-bet controls transcription of two chemokine receptors, CXCR3 and CCR6, which are expressed by GCB cells. Using 50:50 Tbx21 −/−: pepboy chimeras, we tested whether T-bet regulates GCB cell positioning by inducing CXCR3 and repressing CCR6. We observed that the CCR6+ GCB cell subset, which is known to localize in the LZ, was very enriched in Tbx21 −/− GCB cells. By contrast, the CXCR3+ GCB subset, which was dominated by WT cells, was enriched in the DZ. To determine the role for CXCR3 in GCB cell localization, we infected 50:50 Cxcr3 −/− :pepboy chimeras and found that the Cxcr3 −/− GCB cells are enriched in the LZ. Therefore, T-bet regulates GCB cell localization and potentially the dynamics of the GC response by controlling CXCR3 and CCR6 expression.

Mechanisms underpinning poor antibody responses to vaccines in ageing

MYC to BCL6 State-Transitions Determine Cell Size and Metabolic Fluctuations and Define a Novel Biorhythm in B-Cell Malignancies

Peyer's patches of adult mice were morphologically studied by light microscopy.The patch is topographically divided into four portions: germinal center, follicular area, parafollicular area and dome area. The germinal center consists of light and dark zones. The follicular area surrounds the germinal center, especially the light zone which has an apical pole directed toward the mucosal surface. The parafollicular area surrounds the follicular area and contains postcapillary venules with high endothelium. The dome area is situated just above the follicular area. The epithelium over the dome area is infiltrated with numerous lymphocytes.Quantitatively, the germinal center, follicular area and parafollicular area are almost similar in volume (about 30%) and the dome area constitutes only a small portion (about 10%). The density of small lymphocytes is highest in the follicular area and lowest in the germinal center. Out of the total small lymphocytes in the patch, 8.4% are included in the germinal center; 58.2% in the follicular area; 26.7% in the parafollicular area; and 3.8% in the dome area. Intraepithelial small lymphocytes are almost twice as abundant in the dome as in the villi. In the dome, almost half the intraepithelial lymphocytes are above the nuclear level of the epithelial cells, whereas in the villi 7.3% of intraepithelial lymphocytes are above the nuclear level.The results suggest that the functional structure of the Peyer's path is similar to that of peripheral lymphatic tissues.