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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+2) 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.

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 FM 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-mu 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-mu-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-mu 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.

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

We conclude that our data are most consistent with the hypothesis that individual bursal stem cells give rise to multiple clones of B lymphocytes by a preprogrammed sequence of variable (V) region genes. The alternate hypothesis that each stem cell gives rise to one B cell expressing but one set of VH genes could only fit our data if the additional assumption is made that each stem cell is preprogrammed to give rise to an unique B-cell clonotype at a fixed time during development. Since we were unable to influence the pattern of expression of clonotypic diversity by modifying the exposure to antigens and since random somatic mutations would seem an inefficient mechanism for systematic generation of the diversity, we favor the possibility of a genetic mechanism involving an orderly pairing of germ-line VH and VL genes. The corollary of this hypothesis is that V-region diversity is generated first and Ig-class diversity is secondarily expressed within each clone by a switch mechanism for the sequential expression of CH genes.

To gain insight into the prolonged susceptibility to infections noted after allogeneic bone marrow transplantation (BMT), multiple parameters of the humoral immune system were serially monitored in ten bone marrow recipients. IgM B cells appeared in the circulation 2 to 4 mo after engraftment. During the first 6 mo, the IgM B cells expressed low levels of CD21 (C3d/EBV receptors) and were largely CD38+. IgG and IgA B cells were also found to coexpress surface IgM and IgD, indicating that they may be involved in a process of isotype switch. These features are characteristic of neonatal B cells. To explore the pattern of Ig isotype switch, the emergence of plasma cell precursors for each of the four IgG subclasses was examined by culturing blood lymphocytes with PWM or LPS and enumerating bone marrow plasma cells. A marked IgG2 and IgG4 plasma cell deficiency and a relative increase in IgG1 and IgG3 plasma cells were detected both in vitro and in vivo. Serum IgG2 and IgG4 levels were deficient for more than 18 mo after BMT, elevated IgG1 levels accounting for the normal or increased levels of total IgG. The data suggest that a selective unresponsiveness to polysaccharide Ag and IgG2 subclass deficiency may contribute to the late bacterial infections in BMT recipients. These features of gradual development of the humoral immune system in adults undergoing successful marrow engraftment reproduce some of the maturational steps that occur during normal B cell ontogeny over the first 1 to 2 yr of life.

Chromatin fractions (DNA, histones and non-histone chromosomal proteins NHCP) have been isolated from human peripheral B and T lymphocytes using different methods and analyzed in order to identify their lipid content. While DNA and histone fractions do not reveal the presence of lipids, a 2% of phospholipids is present in the NHCP fraction. The phospholipids associated with NHCP present a constant relative ratio among sphingomyelin, phosphatidyl-choline and phosphatidyl-ethanolamine both in B and T lymphocytes, whichever are the extraction procedures employed. These findings are related to the possible dereprssive role of phospholipids on DNA-dependent RNA synthesis.

Circulating malignant cells from 15 patients with B-cell chronic lymphocytic leukemia (B-CLL) were examined by the electron microscope (EM) and tested for their capacity to produce immunoglobulin (Ig) molecules using endogenous labeling techniques in vitro. In agreement with previous observations, B-CLL clones from various patients could be subdivided in three distinct groups: immature (type 1) clones, that comprised mainly small resting lymphocytes which synthesized, but degraded Ig of the secretory type intracellularly; mature (type 3) clones consisting mainly of cells with an extended Golgi apparatus and numerous strands of rough endoplasmic reticulum (RER) that secreted Ig molecules; and clones (type 2) at an intermediate maturational stage. When stimulated with T-cell supernatants that contained T-cell replacing factor(s) (TRF), type 3 and, to a lesser extent, type 2 clones differentiated further and secreted increased amounts of Ig. This maturation was not observed when type 1 clones were stimulated with the same supernatants. However, these cells were not incapable of further maturation since they could differentiate in response to phorbol ester (TPA). The present data reinforce the concept that different CLL clones undergo a process of maturation in vivo that may be arrested at different levels and demonstrate that the various clones respond differently to physiological stimuli depending upon the level of maturation already reached in vivo.

The capacity of synthesizing and secreting Ig molecules was studied in 11 patients with B-cell chronic lymphocytic leukemia (B-CLL) whose cells expressed surface IgM, in 3 patients with surface IgG-bearing cells, and in 2 IgM prolymphocytic leukemias (IgM-PLL). Three types of mu chains were detected by SDS-polyacrylamide gel electrophoresis analysis of the endogenously labeled molecules isolated by specific immunoprecipitation. Two of them were isolated from the cell lysates and were identified as the membrane mu chain and the precursor of the secreted molecules, respectively. The latter also possibly contained precursors of the membrane molecules. The third type of molecule was detected only in the culture medium and was identified as secretory mu chain. Not all of the malignant clones possessed the three types of mu chains. Only 7/13 of the IgM-bearing malignant cell clones were capable of secretion, whereas the remaining synthesized the secretory mu chains but degraded them intracellularly. Two types of molecules (membrane and secreted) were found in the IgG-bearing CLL cells from three patients. In all of them, secretion was detected. Ultrastructural analysis demonstrated that cells from the secreting clones had the features of more mature lymphocytes than the cells from nonsecreting clones. These features were represented by a developed Golgi apparatus, various types of vesicles (smooth and coated), and strands of the rough endoplasmic reticulum. A certain heterogeneity of the degree of maturation of the cells was observed within these clones. The data are consistent with the hypothesis that CLL clones are heterogeneous and can be distinguished through the different degrees of maturation of their cell components.

Subpopulations of thymus-derived T lymphocytes bearing receptors for either IgM or IgG molecules were isolated from human peripheral blood. Those with receptors for IgM (T.M) provided help in a cell dose-dependent fashion for the pokeweed mitogen-induced differentiation of B lymphocytes in vitro, whereas cells with receptors for IgG (T.G) did not. T.G cells, on the hand, efficiently suppressed the differentiation and proliferation of B cells in the pokeweed system in the presence of helper T.M cells. This suppressive activity of T.G cells required prior interaction of the T.G cells with immune complexes. The helper activity of T.M cells was relatively radioresistant while the suppressor activity of T.G cells was radiosensitive. The results indicate that helper and suppressor functions of human T lymphocytes in this model system are mediated by different subpopulations of T cells which can be distinguished by their ability to bind IgM or IgG immune complexes, respectively.

Heterogeneity within lymphocyte subsets expressing T-helper (T4+/Leu3+) or T-suppressor (T8+/Leu2+) markers was analyzed in 38 patients with B cell chronic lymphocytic leukemia (B-CLL) and in 11 age-matched controls. Co-expression of NK-lineage markers (M1, Leu7) on Leu2+ or Leu3+ cells was investigated by two-color immunofluorescence, and the proportion of granular lymphocytes within each subset was determined by cytochemical staining for acid phosphatase. B-CLL patients and normal controls had similar absolute numbers of cells per microL with T-suppressor phenotype. However, the proportion of Leu2+ cells co-expressing the Leu7 antigen was higher in the B-CLL patients than in the control subjects (54 +/- 3% v 27 +/- 4%, P less than .0001). The absolute number per microL of cells with T-helper phenotype was somewhat decreased in B-CLL patients compared with normal subjects (649 +/- 104 v 799 +/- 33, P less than .02), with a consequent decrease of the helper/suppressor ratio. Furthermore, co-expression of the Leu7 and, more strikingly, of the M1 markers was increased significantly on Leu3+ cells from B-CLL patients compared with normal controls (11 +/- 2% v 2 +/- 0.7%, P less than .002 for Leu7 and 40 +/- 5% v 4 +/- 1%, P less than .00001 for M1). Cytochemical studies showed that a large proportion of Leu3+ cells from B-CLL patients were granular lymphocytes, as suggested by the co-expression of natural killer (NK) cell markers. The emergence of a population of Leu3+ granular lymphocytes with NK markers, which is barely detectable in normal subjects, may provide an explanation for the impairment of T cell functions repeatedly described in B-CLL.

The initial development and distribution of lymphocytes expressing surface IgM (sIgM) and of specific antigen-binding cells (ABC) were studied in the chicken in an attempt to gain information on the process by which B-cell diversity is generated. The antigens used were sheep erythrocytes (SE), keyhole limpet hemocyanin (KLH), and poly-L(Tyr, Glu)poly-D,L-Ala-poly-L-Lys (TGAL). The results indicate that generation of the total sIgM-positive population begins in the bursa and that specific clones of ABC develop in a fixed sequential pattern which is not influenced by either deprivation of or deliberate exposure to exogenous antigens. Cells bearing sIgM by immunofluorescence (IgM-positive cells) were detected first in the bursa on the 12th day of incubation, KLH-ABC and TGAL-ABC by the 16th day, and SE-ABC on the 18th day. The doubling time of the sIgM-positive population of bursal cells was determined to be approximately 10 h before significant antigen-independent seeding to the spleen began a few days before hatching. Clonal expansion of SE-ABC in the bursa also appeared to be antigen independent as was the initial development of SE-ABC in the blood and spleen which ceased abruptly after bursectomy at hatching. Specific ABC were observed to develop in multiple bursal follicles as small foci of ABC among the much larger total population of sIgM-positive cells within an individual follicle. Intravenously infused SE-ABC homed to the embryonic spleen but not to the bursa. The results are interpreted as favoring a hypothetical model in which individual stem cells give rise to multiple clones of B cells by a predetermined pattern of sequential expression of variable region genes.

The Leu-2 antigen is expressed on a subpopulation of human T cells that perform suppressor and cytotoxic functions. In addition, this antigen is also present on a portion of cells with morphologic characteristics of granular lymphocytes. Although both Leu-2+ cells and granular lymphocytes have been shown to suppress B cell differentiation, the interrelationship of these two suppressor populations has not previously been fully characterized. We recently produced a monoclonal antibody, termed D12 (anti-Leu-15), which reacts with a variety of cell types, including a subpopulation of Leu-2+ cells. Previous studies have indicated that the Leu-2+ cells that suppress T cell proliferative responses express the Leu-2+15+ phenotype, whereas the precursor and effector cytotoxic T cells that recognize class I major histocompatibility antigens are Leu-2+15- lymphocytes. For this report, we used the anti-Leu-2 and anti-Leu-15 monoclonal antibodies and fluorescence-activated cell sorter techniques to characterize the E+ cells that suppress PWM-induced B cell differentiation. These studies indicate that the vast majority of Leu-2+ cells that suppress this T cell-dependent B cell response have the Leu-2+15+ phenotype. Furthermore, when the morphologic and cytochemical characteristics of these Leu-2+15+ cells were studied, virtually all of these cells were granular lymphocytes. Most of the Leu-2+15+ suppressor cells co-expressed the HNK-1 (Leu-7) antigen, which is detected only on granular lymphocytes. In contrast, virtually none of the Leu-2+15+ granular lymphocytes expressed Fc receptors for IgG molecules. These data indicate that the Leu-2+ cells that suppress PWM-induced B cell differentiation are Leu-2+15+ (and predominantly Leu-7+) granular lymphocytes that do not express Fc receptors. The implications of these observations concerning the relationship of human Leu-2+ suppressor cells to murine Ly-2+ cells and the lineage of granular lymphocytes are discussed.

Malignant lymphocytes from 30 B-cell chronic lymphocytic leukemia (B-CLL) patients were studied for the cytochemical localization of two acid hydrolases, alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AT). The large majority of the cells stained for both ANAE and AP in 7 cases, for AP only in 18 cases, and were negative for both the enzymes in 5 cases. Ultrastructural analysis revealed that the cells that displayed more mature morphological features, such as well developed smooth and rough membrane compartments, were those positive for acid hydrolases. That ANAE and AP are expressed by B cells at late stage of maturation was confirmed by the finding that some lymphocytes and all of the plasmacytoid lymphocytes and plasma cells from Walderström's macroglobulinemia, from mixed cryoglobulinemia, and from multiple myeloma patients stained strongly for both ANAE and AP. Using the expression of acid hydrolases and certain ultrastructural features as markers of cell differentiation, it was possible to demonstrate a process of maturation within the single B-CLL clones with accumulation of the cells at stages that differed in the various cases.