Production of two monoclonal antibodies (FB1 AND FB21) useful for the identification of human B lymphocytes in formalin-fixed,paraffin-embedded tissues

Two monoclonal antibodies (FBI and FB21) reactive in formalin-fixed, paraffin-embedded tissue sections are reported in this paper. FB1 and FB21 recognize a cytoplasmic antigen and a surface antigen of B cells, respectively. FBI reacts with mantle zone (MZ) B cells, germinal centre (GC) cells, and marginal zone (MrZ) B cells, but not with T cells in lymphoid tissues. FB21 reacts with MZ B cells, GC cells in lymphoid tissues, and T cells of peripheral blood, but not with MrZ B cells in the spleen. Neither monoclonal antibody (MoAb) reacts with monocytes, granulocytes, or plasma cells. FB1 reacted with all the B-cell lymphomas tested and with CD20-positive Reed-Sternberg cells in two of five cases of Hodgkin's disease, but not with multiple myelomas or T-cell lymphomas. FB21 reacted with B-cell lymphoma in 20 of 22 cases, but not with multiple myelomas, T-cell lymphomas, or Reed-Sternberg cells of Hodgkin's disease. Immunoprecipitation studies revealed that FB1 recognizes the same two polypeptide chains that are recognized by L26 and is a member of the CD20 antibody cluster. FB21 was thought to recognize a sialic acid-dependent carbohydrate epitope and this was confirmed at the Fifth International Conference on Human Leukocyte Differentiation Antigens (Boston, 1993) FB21 did not react with splenic MrZ B cells and was different from the pan B markers reported previously [CD20 (L26), CD45RA (MB1), and CD74 (LN-2)]. FB21 recognizes a subset of B cells and appears to be closely related to CD75/76 antibodies. FB1 and FB21 are useful MoAbs for the diagnosis and analysis of B-cell lymphomas.     

The expression of the B-cell marker mb-1 (CD79a) in Hodgkin's disease

Recent evidence indicates that membrane-bound immunoglobulin on B lymphocytes is associated with a molecule which comprises the products of the mb-1 and B29 genes. This molecule is a highly specific marker for B-cells, presumably because of its central functional role in antigen triggering, and has recently been clustered as CD79a at the 5th Leucocyte Workshop. Recently there has been controversy surrounding reports of B-cell antigen expression by Reed–Sternberg and related cells, and we have therefore studied 108 cases of Hodgkin's disease immunohistochemically using a novel antibody which detects mb-1 protein in paraffin sections. The results were compared with those achieved using antibody L26 to detect CD20. The mb-1 protein was present in the neoplastic cells in all 14 cases of lymphocyte predominance Hodgkin's disease studied, and CD20 immunoreactivity was also found in seven of the eight cases of this subtype studied. Of the non-lymphocyte predominance cases, 20% (19/94) expressed mb-1 and 30% (20/67) CD20 in the Reed–Sternberg cells, but the cells positive for either of these two markers usually constituted only a very small proportion of the neoplastic population. However, in occasional cases (one of 94 for mb-1 and five of 67 for CD20), more than 50% of the neoplastic cells expressed one or both B-cell antigens. These results confirm the B-cell origin of the neoplastic cells in lymphocyte predominance Hodgkin's disease, but they also indicate that, contrary to our previous study, mb-1 expression may occasionally be found in what appears, on histological grounds, to be other types of Hodgkin's disease.     

Methods in pathology. Identification of T-cell lymphomas in paraffin-embedded tissues using polyclonal anti-CD3 antibody: comparison with frozen section immunophenotyping and genotypic analysis.

While L26 (CD20) is now well established as a B-cell marker of high specificity for use in paraffin-embedded tissues, paraffin-reactive T-cell antibodies (UCHL1, MT1, Leu-22, DF-T1, and MT2) have not shown comparable lineage specificity. A new commercially available polyclonal antibody directed against a synthetic peptide sequence of the CD3 (T-cell) antigen has recently become available for use on paraffin sections. In order to evaluate the utility of this antibody, we studied CD3 expression in conjunction with L26 and leukocyte common antigen (LCA) in 15 T-cell and 20 B-cell non-Hodgkin's lymphomas (NHL), all genotypically confirmed by DNA hybridization and immunophenotyped by immunoperoxidase studies in frozen tissue. Ten of 15 T-cell NHLs (67%) showed unequivocal immunolabeling of neoplastic cells with anti-CD3 in paraffin-embedded tissue. Of the five negative cases, three were lymphoblastic lymphomas, and two were peripheral (postthymic) lymphomas (one anaplastic large cell, Ki-1 positive and one large cell, immunoblastic). CD3 expression was identical in paraffin and cryostat sections (100% concordance). Twenty of 20 B-cell NHLs were positive with L26 and LCA but were negative with anti-CD3. Other neoplasms examined, including three granulocytic sarcomas and 45 nonhematopoietic tumors, were similarly negative with anti-CD3. We conclude that polyclonal anti-CD3 is a sensitive and highly specific T-cell marker in paraffin-embedded tissue and, when used in conjunction with LCA and L26, that it can determine cell lineage in the majority of non-Hodgkin's lymphomas.     

Immunophenotypic analysis of acute lymphoblastic leukemia using routinely processed bone marrow specimens

Monoclonal antibodies have been recently developed that react with antigens expressed on T and B lymphocytes in routinely processed, paraffin-embedded lymphoid tissues. In this study, we assessed bone marrow clot and/or core biopsy sections of 19 cases of acute lymphoblastic leukemia (ALL) using routinely decalcified, B5- or formalin-fixed, paraffin-embedded sections and a panel of monoclonal antibodies, including LN1, LN2, L26, Leu-22, UCHL-1, and LCA. Each case had been previously phenotyped using freshly obtained aspirate material and a standard immunophenotypic protocol. Our results demonstrate the utility of the LN2 antibody in differentiating between precursor B-cell (pre-B) and precursor T-cell ALL. The LN2 antibody stained 11 of 12 cases of pre-B ALL and did not react with any of the seven T-cell ALLs. The other antibodies tested were less helpful. The Leu-22 antibody stained both pre-B and T-cell ALLs, while the results with UCHL-1 revealed peculiar nuclear staining of pre-B and T-cell ALLs; this we attributed to processing artifact. The L26 antibody reacted with only one case of pre-B ALL (also CD20 antigen positive), while the LN1 antibody did not react with any pre-B ALLs. Neither L26 nor LN1 stained any cases of T-cell ALL. The LCA antibody stained in only four (21%) of 19 cases, two pre-B and two T-cell ALLs. The results also suggest that this panel of antibodies may be useful in differentiating ALL from mature B-cell and T-cell lymphomas involving the bone marrow.     

Primary large-cell lymphoma of the thymus: a diffuse B-cell neoplasm presenting as primary mediastinal lymphoma

Primary mediastinal nonlymphoblastic non-Hodgkin's lymphoma (NLNHL) has distinct clinical, histologic (diffuse large-cell morphology, often with sclerosis and clear cytoplasm), and immunohistochemical features (predominantly B-cell lineage, usually immunoglobulin-negative), which suggest origin from a unique B-cell population. The thymus has a resident population of B cells with a unique immunophenotype, and can be involved by primary mediastinal NLNHL, in some cases selectively. Fifteen cases of NLNHL involving the thymus were studied by paraffin-section immunohistochemistry using antibodies to formalin-resistant epitopes of B cells (4KB5 [CD45RA] and L26 [CD20]) and T cells (L60 [CD43] and UCHL1 [CD45RO]). All were diffuse large-cell or immunoblastic lymphomas with sclerosis, and were also similar to primary mediastinal NLNHL in clinical features. Neoplastic cells stained with L26 in all but one case, which stained with 4KB5 and an antibody to a leukocyte-common antigen (PD7/26 [CD45RB]), and were uniformly nonreactive with L60 (with one exception) and UCHL1. Intermingled small lymphocytes were uniformly L26-negative and positive for T-cell markers, even in one case with atypia suggesting a lymphoma of mixed morphology. These findings demonstrate that primary thymic and primary mediastinal NLNHL are similar B-lineage neoplasms, and support previous suggestions that both may originate in thymic B cells.     

Expression of Leu M1 antigen on a monoclonal B cell line established from a patient with rheumatoid arthritis

The purpose of this study is to show that anti-Leu M1 antibody (anti-CD15), which has different staining characteristics in lymphoid and non-lymphoid cells, reacted against the surface antigen of a defined monoclonal B cell line. This antibody recognizes the sugar moiety, lacto-N-fucopentaose (LNF-III), which is linked to the cell membrane protein in several kinds of cells, but not in B cells. However, a human monoclonal B-cell line (TKS-1) which was established from the peripheral blood of a patient with rheumatoid arthritis, expressed the Leu M1 antigen spontaneously. The analysis of surface markers using a fluorescence-activated cell sorter (FACS) has revealed that the surface markers of TKS-1 were anti-mu, delta, kappa, HLA-DR, DQ, Leu 12 (CD19) and Leu M1 (CD15). TKS-1 cells were not reactive with any of the following antibodies: anti-OK M1 (CD11b), Leu M2, Leu M3 (CD14), Leu M4, Leu 1 (CD5), Leu 2 (CD8), Leu 3 (CD4), Leu 4 (CD3), Leu 7 and Leu 11 (CD16). In addition, TKS-1 was positive to Epstein-Barr nuclear antigen, weakly positive to non-specific esterase without staining inhibition by NaF, and negative to peroxidase. TKS-1 cells produced IgM in the culture supernatant and have kappa-light chain rearrangement in its DNA. As shown in other studies, distribution of Leu M1 is very wide. This antigen is not a specific immunodiagnostic marker to distinguish the cell type. We conclude that it is possible to express Leu M1 antigen on the membrane of a B-cell lineage cell.     

Modulation of IgD and CD20 by ligation of CD5 on tonsillar B cells

The CD5 molecule, pan T cell marker, has been known to be expressed on a minor population of B cells, termed B-1 cells. However, the physiological function and pathological role of CD5+B (B-1) cells remain to be fully elucidated in humans. In the present study, we aimed to clarify the significance of CD5 expression on the B lymphocytes in human tonsil. Using flow cytometric analysis by three-colour immunofluorescence staining, we observed a majority of the cell surface CD5-positive (sCD5+) B cells among the sIgD+ B-cell population, as previously described. Contrary to our expectation, approximately half of the sIgD+/sCD5+ B cells expressed CD38 on their cell surface. Furthermore, a small number of sCD5+ were observed in the sIgD- B cell population. The addition of anti-CD5 monoclonal antibody (MoAb) to the culture induced downmodulation of sCD20 and sIgD of the tonsillar B cells, resulting in an increase of sCD38-/sIgD- (memory) B cells during the 10 day culture periods in the CD40/l cell culture system. Our findings suggest that ligation of CD5 might transduce the signal to regulate B cell maturation.     

Synergistic antibody induction by antigen-CD40 ligand fusion protein as improved immunogen

Full scale B-cell activation requires not only B-cell receptor (BCR) engagement with antigen, but also costimulatory signals provided by T helper cells through the CD40-CD40 ligand (CD40L) interaction. It is hypothesized that a fusion protein of an antigen and soluble CD40L (CD40LT) would selectively target the costimulation to antigen-specific B cells, leading to synergy in the antibody response. This hypothesis was investigated by fusing green fluorescence protein (GFP), a generic antigen, with mouse CD40LT. Studies revealed that immunization in mice with the plasmid encoding GFP-CD40LT fusion protein led to synergistic induction of GFP-specific antibodies, while control plasmid(s) for GFP, CD40LT, or GFP plus CD40LT did not. Immunization with a single dose of the fusion protein also provoked a vigorous GFP-specific immunoglobulin G1 antibody response, but not other antibody isotypes. These results suggest that GFP-CD40LT fusion protein induces a GFP-specific B-cell activation and antibody response in an antigen-guided fashion. The potential application of this novel strategy in vaccine development is discussed.     

Triggering of B lymphocytes through CD23: epitope mapping and studies using antibody derivatives indicate an allosteric mechanism of signalling.

By using five monoclonal antibodies in reciprocal cross-locking studies, a minimum of three epitope clusters have been defined for the B-cell restricted, activation-associated CD23 antigen. Two of the five antibodies were capable of replacing low molecular weight B-cell growth factor (BCGF) in B-cell co-stimulation assays. These two antibodies belonged to the same epitope group, while non-stimulatory antibodies fell outside this cluster. By prior coating of activated B lymphocytes at 4 degrees, all five CD23 antibodies interfered with the subsequent uptake of BCGF activity onto the cells. However, only the two stimulatory antibodies were capable of inhibiting the absorption of BCGF completely. From one of these antibodies, F(ab')2 and Fab fragments were generated and both were found to be equivalent to whole antibody in their ability to mimic BCGF. Immobilized antibody, however, failed to stimulate over a wide range of concentrations. These findings demonstrate that the ability of certain CD23 antibodies to deliver a growth-promoting signal to activated B cells is independent not only of the Fc portion of the molecule but also of receptor cross-linking. The latter observation is indicative of an allosteric mechanism of triggering, a notion supported by the epitope specificity of activation through CD23. The findings are discussed in relation to the putative natural ligands for CD23 and the way they may influence B-cell function through this receptor.     

The use of transgenic mice for the production of a human monoclonal antibody specific for human CD69 antigen

The CD69 antigen is the earliest activation marker expressed on leukocyte surfaces after stimulation and it has been correlated with disease state in a variety of inflammatory and autoimmune diseases. We were interested in the generation of a human monoclonal antibody (mAb) against the CD69 antigen. To do this, mice carrying human Ig transgenes (on an inactivated endogenous immunoglobulin H and Igkappa background) were immunized with rat cells transfected with the human CD69 molecule. From over 2000 hybridoma clones generated in different fusions, we were able to obtain a human monoclonal antibody, hAIM-29, which specifically recognizes human CD69 on the surface of activated-human leukocytes. We demonstrate that the antibody is specific for the human CD69 molecule, as shown by double staining with mouse anti-human CD69 antibodies, ELISA, immunoblot and immunoprecipitation studies. Results of additional experiments show that hAIM-29 activates intracellular calcium influx without Ig cross-linking and enhances phorbol myristate acetate-induced cell proliferation in a manner similar to other mouse anti-CD69 antibodies. This report is the first to describe the isolation and characterization of a novel human mAb, hAIM-29, which may have therapeutic potential in diseases associated with the presence of activated cells expressing CD69 antigen.     

Use of the monoclonal antibody WR17, identifying the CD37 gp40-45 Kd antigen complex, in the diagnosis of B-lymphoid malignancy

The distribution of the gp40-45 Kd antigen bound by the WR17 monoclonal antibody of IgG2 subclass in normal lymphoid tissue was characterized by immunohistochemistry and immunofluorescence staining with flow cytometric analysis. The predominant staining pattern observed was characteristic of an anti-pan-B-lymphocyte reagent. Weak reactions were observed by immunofluorescence staining of viable cell suspensions with all neutrophils and T-lymphocytes in some normal donors. In tissue sections, B-lymphocytes were stained and no cross reactions were observed with T-lymphocytes, although macrophages stained in some sections. A range of T- and B-cell malignancies were stained with WR17 and the reactivity compared to that observed with other monoclonal antibodies in the CD19, CD21 and CD22 clusters. All B-non-Hodgkin's lymphomas, B-chronic lymphocytic, prolymphocytic and hairy cell leukaemia cells examined were stained by WR17 in indirect immunofluorescence assays, whilst the T-cell tumours were negative. The same pattern was observed in cryostat sections of malignant tissue and in addition some tissue macrophages expressed the CD37 antigen cytoplasmically. Intra-tumour heterogeneity of staining was observed with all the monoclonal antibodies tested, although overall WR17 consistently stained B-cell tumours even when expression of the CD19 pan-B-lymphocyte antigen could not be detected with some monoclonals. Monoclonal antibodies, such as WR17, within the CD37 cluster and binding to the gp 40-45 Kd molecule, bind to mature B-lymphocytes and identify the majority of B-cell malignancies.     

Immunohistological analysis of the immunoreactivity of normal lymphoid cells and lymphomas with the monoclonal antibody OPD4

OPD4 is a recently described monoclonal antibody that recognizes a fixation-resistant 200 kD antigen restricted to a subset of T cells. Immunolabelling with OPD4 in paraffin sections of normal lymphoid tissues and cases of malignant lymphoma was compared with that of other antibodies in common use, including the T-cell restricted antibodies MT1 and UCHL1 and the B-cell restricted antibodies MB1, F8-11-13, and L26. OPD4 showed similar immunoreactivity to UCHL1 in normal tissues. OPD4 did not stain Reed-Sternberg cells in Hodgkin's disease. In non-Hodgkin's lymphomas, OPD4, like UCHL1, reacted with only 2/22 B-cell lymphomas. OPD4 was, however, less useful as a marker of T-cell lymphomas, staining only 11/32 cases, while UCHL1 stained 22/32 cases. We conclude that OPD4 is not a useful antibody for the routine diagnosis of T-cell lymphoma.     

Expression of the Ig-associated heterodimer (mb-1 and B29) in Hodgkin's disease

Eighty-three cases of Hodgkin's disease were studied immunocytochemically for the presence of the Ig associated heterodimer (mb-1 and B29) which is believed to be a specific pan B-cell marker. These results were compared with those achieved using other B-cell markers against CD19, CD20 and CD22. Although a small number of cases of nodular sclerosis and mixed cellularity subtype showed positivity for CD19, CD20 or CD22, no case showed any reactivity with antibodies against mb-1 or B29. This contrasted markedly with the cases of lymphocyte predominance where all seven cases expressed one or more of the B-cell antigens, with six cases being positive for mb-1. These results confirm previous studies that have suggested lymphocyte-predominance Hodgkin's disease is of B-cell origin and different from the other subtypes. However, they do not provide support for the thesis that these other subtypes may also have a B-cell origin, albeit with a different phenotype to lymphocyte predominance.     

Immunohistochemical investigation of the cross-reactivity of selected cell markers from various species for characterization of lymphatic tissues in the harbour porpoise (Phocoena phocoena).

To facilitate a detailed investigation of cetacean lymphoid organs, 13 canine-, six bovine-, one equine-, one human- and four killer whale-specific monoclonal antibodies (mAbs) directed against cell surface antigens of the haematopoietic system (including CD2, CD4, CD8, CD45R, MHC class II, granulocyte, thrombocyte, pan-T cell and B-cell antigen), as well as a mAb and a polyclonal antibody (pAb) directed against the -peptide of the human CD3 complex, were tested for immunohistochemical cross-reactivity on frozen or formalin-fixed, paraffin wax-embedded lymphatic tissues of harbour porpoises. Eight of 26 mAbs and the pAb showed a specific reaction with harbour porpoise cells. Lymphocytes in T-cell compartments were labelled by the mAb and the pAb directed against the CD3 complex and by two killer whale mAbs specific for CD2 antigen. CD45R, labelled by a killer whale-specific mAb, was strongly expressed on B and weakly on T cells. MHC class II antigen, recognized by killer whale- and bovine-specific mAbs, was expressed on B and T cells. A canine MHC class II-specific mAb recognized an epitope on the surface of antigen-presenting cells and B lymphocytes. An anti-equine-pan-leucocyte marker labelled the majority of cells in B- and T-cell compartments. Thus, with leucocyte antigen markers from various species, it is now possible to determine the phenotype of lymphocytes in normal and diseased lymphoid organs of harbour porpoises.     

Immunophenotyping of non-Hodgkin''s lymphomas using a panel of antibodies on paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.     

Ploidy, proliferative activity, cluster differentiation antigen expression and clinical remission in highgrade non-Hodgkin''s lymphoma

Using a large range of monoclonal antibodies to specific cluster differentiation antigens the phenotypes of a series of high-grade non-Hodgkin's lymphomas of B- and T-cell type were investigated. Cell ploidy and proliferative fraction were assessed by fluorescent staining of DNA and flow cytometry and data on the incidence of complete clinical remission were obtained. With the exception of some lymphoblastic lymphomas, high-grade B-cell lymphomas normally expressed the pan B-cell antigens CD19 and CD22 but only immunoblastic lymphomas consistently expressed the pan B marker CD20. Variable, generally weak expression of CD21 was observed whilst CD23 expression was most prevalent in rapidly proliferative cases and in Burkitt's and centroblastic lymphomas. A rapidly proliferative, multilobated B-cell lymphoma displayed phenotypic properties intermediate between centroblastic and immunoblastic lymphomas. The T-cell lymphomas generally showed low proliferative activity and expression of CD4 prevailed over CD8. Most cases also showed CD2 and CD5 positivity with some also showing CD3 and CD7 expression. Patients with rapidly proliferative diploid or DNA aneuploid tumours obtained complete remission more readily than patients with lowly proliferative diploid tumours. An excess of early deaths occurred among T-cell cases.     

Use of monoclonal antibodies for the typing of malignant lymphomas in routinely processed biopsy samples

Eight antibodies (UCHL1 (CD45RO), MT1 (CD43), MT2 (CD45R), 4KB5 (CD45R), MB1 (CD45R), MB2, L26 (CD20) and LN1 (CDw75)) have been examined for reactivity with routine specimens of normal and hyperplastic lymphoid organs (n = 6), non-Hodgkin's lymphomas (n = 62), Hodgkin's disease (n = 27) and non-lymphoid malignancies (n = 9). In normal and hyperplastic lymphoid organs, UCHL1 and MT1 stained predominantly T cells; 4KB5, MB1, MB2, L26 and LN1 stained predominantly B cells; and MT2 reacted with a subset of B and T cells. The lineage of the neoplastic cells was correctly identified in 24 of 28 (86%) peripheral T-cell lymphomas; and in 31 of 35 (88%) B-cell malignancies. In two cases of lymphocyte-predominant Hodgkin's disease, the Hodgkin's and Reed-Sternberg (H&RS) cells were 4KB5+, L26+ and/or LN1+. The H&Rs cells in nodular sclerosis and mixed cellularity Hodgkin's disease were positive with 4KB5 in 17 of 25 cases. Antibodies UCHL1, MT1, MB1, MB2, L26 and LN1 also labelled some H&RS cells, but in a much smaller proportion of the cases. In three of nine non-lymphoid neoplasms, UCHL1 and MB2 showed a staining of the neoplastic cells, but the staining was cytoplasmic rather than membrane-associated. The remaining antibodies were unreactive with the non-lymphoid malignancies. It is concluded that many non-Hodgkin's lymphomas can be typed in routine specimens, and that antibodies UCHL1, MT1, L26 and LN1 are especially useful in this respect. The antibodies do not provide a means of distinguishing between non-Hodgkin's lymphomas and Hodgkin's disease.     

Immunoarchitecture of normal human bone marrow: a study of frozen and fixed tissue sections.

To provide baseline information on the immunoarchitecture of normal bone marrow, we studied cryostat-cut, frozen, and paraffin-embedded, fixed tissue sections prepared from 21 core biopsies of normal bone marrow obtained during bone marrow harvests for transplantation. A large panel of antibodies was applied that included, for frozen tissue, Leu-6 (CD1), T11 (CD2), Leu-3a (CD4), Leu-1 (CD5), Leu-2a (CD8), J5 (CD10), My7 (CD13), Leu-11 (CD16), B4 (CD19), B1 (CD20), B2 (CD21), Tac (CD25), My9 (CD33), T200 (CD45), NKH-1 (CD56), kappa and lambda chains, beta F1, Ki-67, HLA-DR, TQ1, and keratin, and for fixed tissue, leukocyte common antigen (CD45), L26 (CD20), LN1 (CDw75), LN2 (CD74), LN3, LN4, LN5, MB1 (CD45R), MB2, MT1 (CD43), MT2 (CD45R), UCHL1 (CD45R0), BM1, Ki-1 (CD30), Leu-M1 (CD15), lysozyme, KP1 (CD68), actin, S100, neuron-specific enolase, vimentin, and keratin. On fresh-frozen sections CD19 and CD2 were the most reliable and sensitive markers for B and T cells, staining 5% and 9% of marrow cells, respectively. Immunoglobulins generally showed heavy background staining, which frequently precluded an accurate assessment. The CD4 to CD8 ratio in the bone marrow was reversed from that of peripheral blood. On fixed tissues, leukocyte common antigen was found in 14% of the marrow cells, corresponding roughly to the lymphocyte population. L26, a pan-B-cell marker, stained 3% of the marrow cells. Among the other B-cell markers, LN1 and MB2 stained a large number of cells (40% to 70%), indicating reactivity with cells of the myeloid or erythroid series in addition to lymphocytes. Among the T-cell markers, UCHL1 and MT1 stained 66% and 50% of the cells, respectively, which could be explained by their cross-reactivity with myeloid cells. Nonspecific myelomonocytic markers (Leu-M1, KP1, and lysozyme) also showed reactivity in a high percentage of cells. No particular architectural distribution patterns of B or T lymphocytes were noted in either frozen or fixed bone marrow specimens. The results of this study provide normal baseline data for the immunohistologic application of hematopoietic and lymphoid markers on frozen or fixed bone marrow biopsy specimens.     

Characterization of a bovine leucocyte differentiation antigen of 145,000 MW restricted to B lymphocytes.

A new bovine B-cell differentiation antigen is described that is detected by three monoclonal antibodies (mAb). The antigen is not an immunoglobulin and is precipitated from peripheral B cells as a molecule with an approximate molecular weight (MW) of 120,000 or 145,000 before and after reduction, respectively. Data obtained from two-colour cytofluorimetry and immunohistochemistry confirmed that the antigen was found only on mature B cells and on cells with dendritic morphology in the follicles of the organized lymphoid tissues. Its level of expression is directly correlated with that of IgM on peripheral blood B cells and Theileria parva-transformed B cells. The marker was also expressed on the peripheral cells which expressed surface IgG. Based on the antigen's cellular distribution, biochemistry and histochemistry, it is considered to be analogous to the human CD21 antigen.     

B-cell antigens within normal and activated human T cells

In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed ('occult') CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules.