Rikiya Tsunoda

Isolation and long-term cultivation of human tonsil follicular dendritic cells

SummaryHighly purified follicular dendritic cells (FDC) were isolated from human tonsils and cultivated for up to 150 days. The cell separation method employed produced pure aggregates (FDC-clusters) composed of FDC and germinal center lymphoid cells, useful for the analysis of the relationship between these two cell types and of the behavior of FDC in culture. During the first few days of culture, lymphoid cells located between FDC extensions survived better than those which were free or partly covered by FDC. After 6 days, the lymphoid population degenerated and only the FDC survived. The unique antigenic pattern of FDC (positive for HLA-DR, DRC-1, CD14b, CD21, CD23, CD35) disappeared within a few days of culture. Recombinant interferon-γ ex-erted a positive effect either on retaining HLA-DR expression or on the reexpression of these antigens by FDC. HLA-ABC antigens were traced until the 10th day and desmosomal junctions until the 14th day. Subsequently, FDC presented peculiar features, including oval and rhomboid shapes, one to ten nuclei, fine amoeboid extensions, stress fibers and a radical dense zone in their cytoplasm. FDC possessed actin, tubulin and vimentin, but neither desmin nor cytokeratin. After 40 days of culture, FDC enlarged and were covered with abundant membrane extensions. Even when kept as long as 150 days in vitro, FDC did not proliferate in any of the culture conditions employed.

An Ultrastructural Study with Enzyme-Labeled Antibody Technique on Immunoglobulin-Containing Cells in Human Tonsils,Especially in Germinal Centers

Localization of IgG, IgA and IgM in human palatine tonsils, especially in germinal centers, was studied with the electron microscopical enzyme‐labeled antibody method. The large germinal center cells differentiate into two kinds of cells within the germinal center; one was the medium‐sized germinal center cells not engaging in intracytoplasmic production of immunoglobulins and another was the immature cells producing at least one of the three classes of immunoglobulins, especially IgM. The latter continued to maturate and developed into the intermediate‐matured cells and probably into the plasmocytes. The three classes of immunoglobulins were also deposited in the form of admixtures in the intercellular spaces among the constituent cells of the germinal centers, mainly attaching on the cell membrane of desmo‐dendric cells. In addition, some of the deposits were found freely in the intercellular spaces. Some demureness between the immunoglobulin‐containing cells outside and within the germinal centers were pointed out.

INNUNOCYTOLOGICAL STUDIES ON THE CONSTITUENT CELLS OF THE SECONDARY NODULES IN HUMAN TONSILS

The rate of presence of surface immunoglobulins (sIg) and incidence of the surface receptors (SRBC‐receptor, Fc‐receptor and C3 receptor) were examined on the constituent cells of secondary nodules enucleated from human tonsils and foated in suspension. As most of the rosette‐forming cells for SRBG‐receptor were judged to belong to small round cells, the germinal center was considered to be a “non‐T‐cell region.” The coronal B‐lymphocytes and small germinal center cells were fairly matured B‐cells because both bore sIg (100%), Fc receptor (25%) and C3 receptor (90‐80%), while the former cells were thought to be more matured on the basis of their possession of surface δ‐chain. As the large germinal center cells bore sig (50%) and carried Fc receptor (25%) and C3 receptor (50%), they seemed to be the major immature cells among the cell constituents of the secondary nodules. Moreover, it is presumed that the majority of the large germinal center cells are able to differentiate into coronal B‐lymphocytes, probably memory cells, and a minor population of them into cIg‐containing cells.

A LIGHT MICROSCOPICAL STUDY OF ISOLATED FOLLICULAR DENDRITIC CELL‐CLUSTERS IN HUMAN TONSILS

In order to re‐examine the cellular structure of isolated FDC‐culsters in the germinal centers of human tonsils, a light microscopical analysis was made. Approximately 14 FDC‐clusters were recovered from one enucleated germinal center using the enzyme digestion technique. The minimum unit of the FDC‐clusters was composed of one FDC and 8 to 9 lymphocytes. Most of the FDC‐clusters were representative of the microenvironment of the light zone at the germinal center in situ. Half of the engulfed centrocytes were supposed to be at the G0 phase, and the others at the G, to G2 phase. It is suspected that the helper‐T cell has some relationship to the FDC microenvironment, and that the suppressor‐T cell does not. Most of the CIgG‐containing cells in the germinal centers were considered to have infiltrated into the interspace of the FDC microenvironment.

Cytokines produced in lymph follicles.

The events occurring inside lymph follicles during a germinal center reaction are poorly understood. Using B and T lymphoid cell populations prepared from human tonsillar lymph follicles, and enriched or not in macrophages or in follicular dendritic cells, we examined the production of cytokines by these cells in vitro. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were found in the supernatants of cultures stimulated with phytohemagglutinin or pokeweed mitogen. IL-1 beta was occasionally detected; its secretion apparently depends on the origin of the tonsils, the stimulation, and the cell populations. IFN-gamma and IL-2 were not produced in significant amounts by these lymph follicle cells. IL-4 was only found in very low concentrations in the supernatant of the different cell cultures. The cell populations containing follicular dendritic cells produced more IL-6 and TNF than the others, especially than those composed of only B and T cells.

Ultrastructural localization of immunoglobulins in hairy cell leukemia

Neoplastic cells from 13 cases of hairy cell leukemia were investigated for immunoglobulin production and lysozyme activity by an electron-immunoperoxidase technique. In 10 cases cytoplasmic immunoglobulins were found, but lysozyme activity was absent in all cases. Immunoglobulins were detected in the perinuclear space and endoplasmic reticulum and at the surface of hairy cells. Of the cases in which immunoglobulins were detected in hairy cells, nine were positive with IgM antiserum and one with IgG antiserum. The immunoglobulins were monoclonal in all cases; six were positive with lambda antiserum and three with kappa antiserum. The class and type of surface immunoglobulins were identical to those of cytoplasmic immunoglobulins in the hairy cells. These results support the conclusion that hairy cells are commonly derived from immunoglobulin-producing B cells at an earlier stage of differentiation than plasma cells.

Immunocytological characterization of the constituent cells of the secondary nodules in human tonsils - II.

Analysis of the morphological and immunocytological characteristics of the germinal center cells in vivo gives a clue to the question of what germinal centers really do. In a previous work (1), the relationship of germinal centers to the possession of B-cell surface characteristics (SIg, Fc receptor & C3 receptor) and the functional differentiation of the germinal center cells were investigated using human tonsils.

DICER1 hotspot mutations in ovarian Sertoli-Leydig cell tumors: a potential association with androgenic effects

Sertoli-Leydig cell tumors (SLCTs) are representative of androgenic ovarian tumors, and they show diverse histologic differentiation, including heterologous differentiation. Genetically, SLCTs are characterized by the presence of DICER1 mutations. In the present study, we analyzed the correlation between somatic DICER1 hotspot mutations and clinicopathological features in 10 ovarian SLCTs. Six of the 10 (60%) SLCTs harbored a DICER1 hotspot mutation. Five of the 6 DICER1-mutated SLCT patients showed androgenic manifestations, including amenorrhea and hirsutism, and 4 of the 6 were associated with the significant elevation of serum testosterone. In contrast, none of the 4 DICER1 wild-type SLCT patients showed virilization. The patient age at diagnosis was lower in those with DICER1-mutated SLCTs (average, 24.7; range, 17-43) than in those with DICER1 wild-type tumors (average, 64.8; range, 47-77). Histologically, heterologous differentiation was found in 4 SLCTs, all of which were DICER1 mutant. Heterologous components included gastrointestinal-type mucinous epithelium (n=3), carcinoid (n=1), and rhabdomyosarcoma (n=1). In the latter, the rhabdomyosarcomatous component was dominant to the SLCT component. In summary, DICER1 hotspot mutations are closely associated with androgenic effects in ovarian SLCTs. It is suggested that DICER1 mutations are involved in the dysregulation of sex hormone synthesis in SLCT patients. Somatic DICER1 hotspot mutations are more common in SLCT patients during the reproductive years than in those during the postreproductive years. DICER1 hotspot mutations may support the pathological diagnosis of SLCTs in cases wherein the heterologous component overwhelms and masks the SLCT component.