Subepithelial B cells in the human palatine tonsil. I. Morphologic,cytochemical and phenotypic characterization

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5⁻ B cells had a surface phenotype (IgM⁺, IgD⁺, CD23⁻, CD38^±, CD10⁻, CD44⁺) that was different from that of FM (IgM⁺, IgD⁺, CD23⁺, CD39⁺, CD38⁻, CD10⁻, CD44^±) and of germinal center (GC) (CD23⁻, CD39⁻, CD38⁺, CD10⁺, CD44^±, IgG⁺) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5⁻ B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5⁻ B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5⁻ B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5⁻ B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP⁺, while GC cells were consistently ALP⁻. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5⁻ B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5⁻ B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.     

Subepithelial B cells in the human palatine tonsil. II. Functional characterization

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike GC B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-μ antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-μ-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-μ antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.     

Only a small proportion of splenic B cells in adults are short-lived virgin cells

A cohort of newly produced virgin B cells was followed from the marrow to the spleen of non-immunized clean rats, which showed minimal antigen-driven proliferation of B cells in their spleens. The progenitors of this cohort of virgin cells were labeled in vivo over 12 h with the thymidine analogue 5-bromo-2′-deoxyuridine (BrdUrd) and their non proliferating progeny left the marrow 2-3 days later. This coincided with the arrival of labeled B cells in the red pulp and T zones of the spleen. These appear to be short-lived as few remained a week after the label was given. The short-lived newly produced virgin B cells can only comprise a minority of splenic B cells, for it is shown that only 20% of splenic B cells are found in the red pulp and T zone. It is calculated that newly produced virgin B cells are likely to make up between 5% and 10% of splenic B cells. In the marginal zones and follicular mantle respective medians of 3.3% and 1.8% were already labeled at 1 day from the start of the BrdUrd pulse. The appearance of these cells seems likely to result from antigen-driven B cell proliferation outside the marrow, for labeled virgin B cells have not started to leave the marrow at this stage. During day 2 and 3 the proportion of labeled follicular mantle B cells rose to 3.4%, which might in part reflect the recruitment of newly produced virgin B cells to the pool of recirculating follicular B cells. After day 3 in the follicles and day 1 in the marginal zones the proportion of labeled cells did not vary significantly through day 7. This appears to confirm the comparative longevity of the cells in these zones, which contain 80% of the non-proliferating splenic B cells of adult rats.     

PMA-activation of peripheral blood and tonsillar B lymphocytes induces large adhesive cells reminiscent of large extrafollicular (monocytoid) B cells

Extrafollicular (EF) B lymphocytes differ in size and morphology depending on the lymphatic organ involved and the kind of inflammatory reaction. On reevaluating EF B cells in various sites and conditions we discriminated three forms: a small (lymphoid) and intermediate (centrocytoid), and a large (monocytoid) variant. Immunohistochemically, these variants could be discriminated by their differential expression of adhesion molecules CD62L (L-selectin) and CD11c: small EF B cells were strongly L-selectin⁺ and CD11c^–; intermediate cells were moderately CD62L⁺ and CD11c^–; large cells were faintly CD62L⁺ or ^– but expressed CD11c. In 72 h cultures of normal peripheral and tonsillar B cells, cross-linking surface immunoglobulin in the presence of interleukin-2 or interleukin-4 led to formation of clusters in vitro together with an increase in cell size and a slight up-regulation of CD11c, as determined by flow cytometry. Stimulation with phorbol 12-myristate 13-acetate (PMA), however, gave rise to large, plastic adherent cells which also showed strong homotypic adhesion, expressed CD62L at minimal levels and CD11c at comparably highest levels and altogether mimicked the large cell variant of EF B cells. We conclude that EF B cells are subjected to cytokine-induced metamorphosis and that differences in cell size and morphology reflect their state of activation and activation-associated adhesion properties. Our data suggest that EF B cells in all anatomical sites are functionally closely related cells which — possibly mediated by CD11c/CD18 — may become sessile and proliferate locally once activated by appropriate signals.     

A case of chromophobe renal cell carcinoma with sarcomatoid foci and a small daughter lesion

Chromophobe renal cell carcinoma (RCC), which has recently been recognized to be a distinct type of RCC, comprises approximately 5% of renal cell tumors. It has been hypothesized that the cells that comprise chromophobe RCC show a phenotype similar to that of intercalated cells of the collecting duct Although the number of research reports on this type of tumor has recently been increasing, only nine cases of chromophobe RCC showing sarcomatoid transformation have been described. Herein, a case of chromophobe RCC with sarcomatoid foci and a small daughter lesion is reported.     

Human circulating specific antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers

Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and α4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.     

Co-ligation of mouse complement receptors 1 and 2 with surface IgM rescues splenic B cells and WEHI-231 cells from anti-surface IgM-induced apoptosis

Recent studies have shown that complement receptors play important roles in both T-dependent and T-independent B lymphocyte responses to low doses of antigen (Ag) in vivo. Complement activation by either the classical or alternative pathway results in the covalent binding of C3 molecules to Ag in forms that ligate complement receptors type 1 (CR1) and 2 (CR2). We hypothesized that C3-bound Ag might cross-link CR2 and/or CR1 with surface (s)IgM and alter the signal that would be transduced through sIgM by Ag binding alone. One result of the altered signal could be the rescue of B lymphocytes from apoptosis that would otherwise be induced by the binding of certain types of Ag alone. We find that co-cross-linking of mouse CR2 and CR1 with sIgM rescues both resting B cells and WEHI-231.7 cells from apoptosis induced by sIgM ligation in a fashion similar to that found using soluble mouse CD40 ligand (mCD40L). Anti-CR2/CR1-mediated rescue requires co-cross-linking of the receptors with sIgM, and has an additive effect on mCD40L-mediated apoptosis rescue. Based on these results, it is likely that the CR2/CR1-derived signal is cooperative with T cell-derived signals such as CD40L and interleukiin-4.     

Characterization of splenic CD21hi T2 B cells

B cell development is a highly regulated process that initiates in the bone marrow (BM) of adult mice. After reaching the IgM⁺ immature stage in the BM, these B cells migrate to the spleen to complete maturation and incorporation into the long-lived peripheral lymphocyte pool. Studies have identified these splenic immature B cells, and have further attempted to delineate the sequence whereby they transition into mature B cells. As such, these immature splenic populations are termed transitional B cells and have been the focus of intense study. The review summarizes the phenotype and currently known functions of the four putative transitional B cell subsets identified to date. Although most appear to represent short-lived transitional B cells, the CD21^hi T2 B cell population exhibits a number of qualities that question its label as a transitional B cell subset.     

Reasons why DBA/2 mice are resistant to malarial infection: expansion of CD3int B220+ gammadelta T cells with double-negative CD4- CD8- phenotype in the liver

DBA/2 (H-2(d)) mice are known to be more resistant than C57BL/6 (B6, H-2(b)) mice to the non-lethal 17XNL strain of Plasmodium yoelii. This is a very strange phenomenon because the functions of conventional T cells, especially CD8(+) T cells, are known to be somewhat lower in DBA/2 mice than in other strains of mice. We examined herein how immune responses differed between DBA/2 mice and B6 mice during malarial infection. DBA/2 mice and (DBA/2 x B6)F(1) (BDF(1), H-2(b/d)) mice were found to have milder parasitaemia and to recover more quickly from malarial infection than B6 mice. These DBA/2 and BDF(1) mice were also found to experience a marked expansion of interleukin (IL)-2Rbeta(+) CD3(int) cells and gammadelta T cells in the liver, especially in the recovery phase. The expansion of unconventional T cells (i.e. B220(+) T cells) was also marked in DBA/2 and BDF(1) mice. The majority of B220(+) T cells were gammadelta T cells and these T cells were double-negative CD4(-) CD8(-). More importantly, the production of immunoglobulin M (IgM)-type anti-DNA autoantibody was also higher in DBA/2 and BDF(1) mice than in B6 mice. In conjunction with data on cytokine production, these results indicate that primitive T and B cells, namely autoreactive extrathymic T cells and autoantibody-producing B cells, may be much more activated in DBA/2 mice and therefore resistant to the non-lethal 17XNL strain of P. yoelii.     

Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa

Aberrant crypt foci (ACF) in colorectal mucosa are proposed to be the earliest morphological lesion in the development of neoplasia, but their characteristics remain controversial. We therefore studied the epithelial phenotype and genotype of ACF from patients with familial adenomatous polyposis (FAP) and of sporadic ACF by evaluating glycoprotein markers associated with neoplasia (lectins Dolichus biflorus agglutinin and peanut agglutinin; monoclonal antibody CA 19-9 against sialyl Lewis-a blood group substance), expression of proliferating cell nuclear antigen, and ras proto-oncogene mutations. The utility of the markers was established by comparing adenomas and hyperplastic polyps. Most FAP ACF resembled adenomas and were found to differ from sporadic ACF in their high frequency of dysplasia, staining with Dolichus biflorus agglutinin, expression of sialyl Lewis-a, proliferation in the epithelium of upper crypts, and low frequency of ras gene mutations (P = . 04 to <. 0000001). By contrast, sporadic ACF and a subset of FAP ACF had phenotypic characteristics resembling hyperplastic polyps but usually had ras mutations, which were inversely related to dysplasia (P = . 00009). Our findings suggest that “aberrant crypt focus” is a generic term analogous to “polyp” and requires further histopathologic, phenotypic, or genotypic classification into dysplastic and heteroplastic (hetero = other, PLASIA = form) types. Dysplastic ACF represent potential precursors to colorectal adenomas and adenocarcinomas, but heteroplastic ACF appear to be associated, rather than precursor, lesions.     

The CD45 isoform B220 identifies select subsets of human B cells and B-cell lymphoproliferative disorders

The B220 isoform of CD45, a pan B-cell marker in mice, is expressed by only a subset of human B cells that do not express the memory B-cell marker CD27, suggesting that it is a differentiation-specific isoform of CD45. We examined normal human peripheral blood B cells, secondary lymphoid tissue, and a range of human B-cell lymphoproliferative disorders for the expression of B220 by flow cytometric immunophenotyping and immunohistochemical staining. We found that a subset of human B cells in peripheral blood is positive for B220 by flow cytometric immunophenotypic analysis. In reactive lymphoid tissues, B220 is expressed by B cells occupying the mantle zones and by a subpopulation of germinal center cells, but, in contrast, marginal zone B cells in the spleen do not express B220. Of 94 cases of B-cell lymphoproliferative disorders, 33 (35%) were positive for B220 by flow cytometric immunophenotypic analysis, including most cases of marginal zone lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. In contrast, all cases of precursor B lymphoblastic leukemia/lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma were negative for B220. Immunohistochemical staining for B220 correlated with flow cytometric analysis for all cases studied by both methods. Our data demonstrate that B220 is expressed in a select subset of normal, reactive B cells in a pattern that is consistent with a marker of naive B cells. However, this restricted expression pattern is not seen in B-cell lymphoproliferative disorders. Discordance between the B220 expression patterns of normal mantle and marginal zone B cells and their respective neoplastic counterparts may aid in the distinction between normal and neoplastic proliferations at these anatomical sites.     

In vivo treatment with anti B-220 monoclonal antibody affects T and B cell differentiation.

The B220 cell marker is expressed on B cells and on T cell precursors. In order to determine the involvement of the B220 antigen on murine lymphoid differentiation, we treated 5-10-week-old mice periodically with a specific anti-B220 antibody, RA3-6B2, a non-cytolytic IgG2b. After the third injection, a significant reduction (P less than 0.02) in the number of thymocytes and less dramatically in the number of splenocytes was observed. This reduction was predominantly due to a decrease of cells carrying the following markers: Thy-1.2+, Lyt-1+, Lyt-2.3+, L3T4+, and asGM1+. Mitogenic response to concanavalin A, phytohaemagglutinin and lipopolysaccharide, mixed lymphocyte reaction, cytotoxic T cell activity, and plaque-forming cell generation were significantly decreased after the treatment (P less than 0.01). These results show that the in vivo treatment with anti-B220 monoclonal antibody reduced the number of T and B cells and modified their functional activity. This suggests that the B220 antigen is involved in the maturation of both T and B cells.     

The fate of adoptively transferred antigen-specific T cells in vivo

In this study, anti-major histocompatibility complex class I L^d T cell receptor (TCR)-transgenic cells were adoptively transferred into severe-combined immunodeficient (Scid) mice which express the L^d Ag on all nucleated cells, and the fate of transferred antigen-specific T cells (ASTC) was followed in vivo. It was found that, after encountering antigen (Ag) in vivo, the number of ASTC increased 10–15-fold followed by a decline in number to a value that was still above the starting value. The expansion of ASTC could be abrogated by blocking CD28 on the T cells prior to Ag stimulation. Using the technique which simultaneously stains ASTC surface markers and apoptotic cells, we have not only demonstrated directly that the ASTC that disappeared from the periphery died by activation-induced apoptosis but also studied their kinetics in vivo. The remaining ASTC moderately down-regulated both TCR and CD8 on their cell surface and were fully unresponsive when cultured with L^d+ cells even in the presence of exogenous interleukin (IL)-2 and IL-4. However, they were still susceptible to apoptosis when transfereed into a secondary host that provided a new source of Ag and antigen-presenting cells. These studies indicate that peripheral T cell tolerance can be induced by multiple mechanisms in which activation-induced ASTC apoptosis plays an important role.     

Quiescent memory B cells in human peripheral blood co-express bcl-2 and bcl-xL anti-apoptotic proteins at high levels

The anti-apoptotic proteins bcl-2 and bcl-x_L seem to exhibit strictly opposite expression patterns in normal lymphoid cell differentiation stages, with bcl-2 low and bxl-x_L high in immature and mature proliferating cells, the reverse being the case in recirculating quiescent cells. However, it is in fact not known whether recirculating memory cells are bcl-x_L low or high. We analyzed memory (immunoglobulin isotype-switched) B cells in human peripheral blood, which were small lymphocytes in the G0 phase of the cell cycle, but proliferated better than naive B cells in response to Staphylococcus aureus Cowan I. Ex vivo these cells co-expressed bcl-2 together with bcl-x_L mRNA and protein at high levels. The mcl-1 mRNA level was low. The bcl-x_L mRNA level decreased during culture in medium containing fetal calf serum, which implies that it is maintained in vivo by continuous or frequent, non-mitogenic signal(s). The high bcl-x_L expression of memory B cells may be relevant with regard to their longevity and/or their capacity to undergo an accelerated secondary type immune response.     

Characterization of hyperplastic foci observed in surgical specimens of hepatocellular carcinoma

By reviewing previous surgical specimens of hepatocellular carcinoma, 17 cases with hyperplastic foci (HPF) characterized by discernible increase in nuclear densities, could be histologically selected. Nuclear densities of HPF and control hepatic parenchyma were assessed quantitatively by counting the nuclear number of hepatic cells, and proliferating cell nuclear antigen labeling index was measured. HPF occurred multifocally, confined within a lobular unit, smoothly merging into surrounding hepatic parenchyma. Nuclear densities of HPF were 1.71 times greater than those of control hepatic parenchyma. The hepatocytes of HPF also showed significantly higher proliferative activities than those of control parenchyma. In addition, noticeable structural distortions, such as focal trabecular thickening or microacinar formation of hepatocytes, were sometimes observed in HPF. However, these HPF seemed to be distinguished from minute de novo hepatocellular carcinoma (HCC) or intrahepatic HCC metastasis, because of paucity of distinctive atypical changes, and intimate correlation with neighboring hepatocytes. Several adjacent HPF were aggregated to form a much larger unit of a hyperplastic area with loss of fibrous septa of liver cirrhosis. It was suggested that grossly detectable large regenerative nodules are produced via fusion of several adjacent HPF.     

The role of bcl-xL in CD40-mediated rescue from anti-μ-induced apoptosis in WEHI-231 B lymphoma cells

The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2-independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-x_L) renders these cells refractory to anti-Ig-induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-x_L protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-x_L. We, therefore, conclude that bcl-x_L plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype.     

Generation of B220low B cells and production of autoantibodies in mice with experimental amyloidosis: association of primordial T cells with this phenomenon

To investigate the immunological state in amyloidosis, mice were twice intraperitoneally injected (2-week interval) with casein emulsified in complete Freund's adjuvant. Two weeks after the treatment, amyloid deposits were detected in the spleen and other organs of these mice. The number of lymphocytes yielded by the liver and spleen increased significantly. The most affected lymphocyte subset was found to be B cells, namely, the total number of B cells increased and unusual B220low B cells were newly generated in the liver and spleen. In other words, not only normal B220high B cells but also unusual B220low B cells were detected in these organs of mice with amyloidosis. In parallel with this phenomenon, autoantibodies against denatured DNA were detected in sera. Since such autoantibodies are known to accompany the functional activation of NKT cells, NKT cell-deficient mice were used for the induction of amyloidosis. Such mice showed less formation of amyloidosis and lower levels of autoantibodies in sera. Athymic nude mice were NKT cell-deficient but NK1.1- TCRint cells were present. These athymic mice showed an intermediate induction of amyloidosis. The cytokine profile seen in mice with amyloidosis was the Th0 type, showing simultaneous production of IL-4 and IFNgamma. These results suggest that the generation of B220low B cells and the production of autoantibodies in aid of primordial T cells may be major immunological mechanisms in amyloidosis mice.