Immunoreactivity of B-cell markers (CD79a, L26) in rare cases of extranodal cytotoxic peripheral T- (NK/T-) cell lymphomas.

The monoclonal antibodies L26 (CD20) and CD79a are very useful reagents for the immunohistochemical assessment of B-cell lineage in lymphoproliferative disorders. Although very few CD20-positive peripheral T-cell lymphomas (PTL) have been reported, comprehensive analyses of CD79a reactivity in extranodal PTL and NK/T-cell lymphomas have not been performed previously. This study investigated CD79a (clone JCB117) and CD20 reactivity in 94 extranodal non-B-cell lymphomas (enteropathy-type intestinal T-cell lymphoma [n = 52], nasal NK/T-cell lymphoma [n = 11], and primary cutaneous PTL [n = 31]) and in 17 cases of nodal PTL, unspecified. In four cases (enteropathy-type intestinal T-cell lymphoma [n = 3] and nasal NK/T-cell lymphoma [n = 1]), the majority of tumor cells stained for CD79a (all CD20 negative) and one cutaneous PTL, unspecified, was CD20 positive (CD79a negative). Extensive immunophenotyping and polymerase chain reaction-based molecular analyses revealed that all five B-cell marker-positive extranodal lymphomas had a cytotoxic phenotype and did indeed represent monoclonal peripheral T-cell proliferations. To minimize the risk of misinterpretation of lymphoma cell lineage, especially in cases of extranodal, lymphoproliferative disease, we suggest the use of both CD79a and CD20 in combination with a panel of antibodies reactive to T cells, such as betaF1 and CD5, and to T cells and NK cells, such as CD3, CD2, CD56, and TIA-1.     

CD23 expression in mediastinal large B-cell lymphomas

AIMS: Mediastinal large B-cell lymphoma (MLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) in the WHO classification with peculiar features, such as female prevalence, young patient age and bulky presentation. It shows a B-cell phenotype with variable expression of surface immunoglobulin, negative CD21 and CD10 and positive CD30 in a large number of cases. An origin from activated thymic B cells has been suggested in several studies. A subpopulation of large, dendritic cells (asteroid cells) strongly expressing CD23 has been identified amongst thymic B cells and these could represent the normal cellular counterpart for this type of primary mediastinal large cell lymphoma. METHODS AND RESULTS: To explore this possibility, we immunostained 24 cases of primary mediastinal lymphomas and 100 cases of non-mediastinal, nodal and extranodal, DLBCLs for CD23 in routinely processed paraffin-embedded tissues. CONCLUSIONS: Our results show that a vast majority (70%) of mediastinal lymphomas strongly express CD23 whilst the same antigen is expressed in only 15% of non-mediastinal nodal DLBCLs and 9% of non-mediastinal extranodal DLBCLs. These results support the hypothesis that most cases of MLBCL arise from activated dendritic thymic B cells. We also suggest that CD23 should be included in the panel of antibodies currently used to characterize this subtype of DLBCL.     

Lineage determination of CD20- B-Cell neoplasms: an immunohistochemical study

We studied 61 CD20- B-cell lymphomas, including 29 cases of precursor B-cell lymphoblastic leukemia/lymphoblastic lymphoma (B-ALL/B-LBL), 25 cases of CD20- recurrent mature B-cell lymphoma after rituximab therapy, and 7 cases of CD20- diffuse large B cell lymphoma (DLBCL). We used markers specific for B lineage: CD79a, Pax-5, OCT.2, and BOB.1. All B-ALL/B-LBLs expressed Pax-5 (29/29 [100%]), 25 (93%) of 27 expressed BOB.1, 23 (79%) of 29 expressed CD79a, and 6 (22%) of 27 expressed OCT.2. The percentages of cases expressing Pax-5, CD79a, OCT.2, and BOB.1 in CD20- recurrent mature B-cell lymphomas after rituximab treatment were 88% (21/24), 84% (21/25), 81% (17/21), and 73% (16/22), respectively. CD20- DLBCLs rarely express routine B-lineage markers, such as and CD79a and Pax-5, but they expressed OCT.2 or BOB.1. Pax-5, BOB.1, and CD79a antigens are the most reliable B-lineage markers for paraffin immunophenotyping B-ALL/B-LBL. CD79a and Pax-5 should be used as the first-line B lineage-specific markers for rituximab-treated CD20- mature B-cell lymphomas. If negative, OCT.2 or BOB.1 may be useful. The newly identified B-lineage markers, OCT.2 and BOB.1, may be the most useful for the B-lineage determination of CD20- plasmablastic or primary effusion subtypes of DLBCL.     

Both CD4+ FoxP3+ and CD4+ FoxP3- T cells from patients with B-cell malignancy express cytolytic markers and kill autologous leukaemic B cells in vitro

Cytotoxic CD4(+) T cells have been found in patients with chronic lymphocytic leukaemia (CLL) and seem to be involved in the regulation of malignant B cells. The CD4(+) T regulatory cells (Tregs) can regulate various immune cells, including B cells, by inducing their apoptosis. Hence, different subgroups of CD4(+) T cells may be involved in the regulation of malignant B cells. In this study, the cytotoxic phenotype and function of various CD4(+) T-cell subgroups were investigated in patients with B-cell malignancies. Peripheral blood was collected from patients with CLL, various B-cell lymphomas, healthy adult donors, children with precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) and from healthy children. CD4(+) T cells (CD3(+) CD4(+) FoxP3(-)), Tregs (CD3(+) CD4(+) CD127(low) FoxP3(+)) and CD127(high) FoxP3(+) T cells (CD3(+) CD4(+) CD127(high) FoxP3(+)) were analysed for their expression of the cytolytic markers CD107a and Fas ligand. Patients with CLL had increased CD107a expression on all tested T-cell subgroups compared with healthy donors. Similar results were found in patients with B-cell lymphomas whereas the CD107a expression in children with pre-B ALL was no different from that in healthy controls. Fas ligand expression was similar between patient cells and cells of healthy donors. CD4(+) T cells and Tregs from patients with CLL and healthy donors were subsequently purified and cultured in vitro with autologous B cells. Both subgroups lysed B cells and killing was confirmed by granzyme ELISAs. In conclusion, cytotoxic populations of CD4(+) T cells, including Tregs, are present in patients with B-cell malignancy and may be an important factor in immune-related disease control.     

Diagnostic importance of CD179a/b as markers of precursor B-cell lymphoblastic lymphoma.

Surrogate light chains consisting of VpreB (CD179a) and lambda5 (CD179b) are expressed in precursor B cells lacking a complete form of immunoglobulin and are thought to act as substitutes for conventional light chains. Upon differentiation to immature and mature B cells, CD179a/b disappear and are replaced with conventional light chains. Thus, these molecules may be useful as essential markers of precursor B cells. To examine the expression of the surrogate light-chain components CD179a and CD179b in precursor B-cell lymphoblastic lymphoma, we analyzed tissue sections using immunohistochemistry techniques. Among a number of monoclonal antibodies for the surrogate light chains, VpreB8 and SL11 were found to detect CD179a and CD179b, respectively, in acetone-fixed fresh frozen sections. Moreover, we also observed VpreB8 staining in formalin-fixed, paraffin-embedded sections. Using these antibodies, we found that CD179a/b were specifically expressed in precursor B-cell lymphoblastic lymphomas, but not in mature B-cell lymphomas in childhood. Furthermore, other pediatric tumors that must be included in a differential diagnosis of precursor B-cell lymphoblastic lymphoma, including precursor T-cell lymphoblastic lymphoma, extramedullary myeloid tumors, and Ewing sarcoma, were also negative for both CD179a and CD179b. Our data indicate that CD179a and CD179b may be important markers for the immunophenotypic diagnosis of precursor B-cell lymphoblastic lymphomas.     

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.     

The MHC class I associated beta 2-microglobulin (beta 2m) light chain is expressed in a molar excess over HLA-ABC and CD1 on the membrane of leukaemic B cells but not leukaemic T cells: evidence for further beta 2m-associated molecules.

Beta 2-microglobulin (beta 2m) constitutes the common light chain of both the MHC-encoded HLA-ABC molecules and a group of structurally related glycoproteins recognized by antibodies of the first cluster of differentiation (CD1a, CD1b and CD1c). These CD1 antigens appear similar to murine T1 and Qa molecules in terms of structure and tissue distribution, although the question of inter-species homology is controversial. A further group of alloantigens expressed predominantly on T cells has been reported however, with immunogenetic characteristics more closely analogous to the murine T1/Qa system than the CD1 antigens, although their precise identity remains ill-defined. Having previously shown that malignant B cells may express membrane CD1c, we examined leukaemic B-cells corresponding to early lymphoblastic differentiation (null- and common acute lymphoblastic leukaemia) through to the terminal plasma cell stage for the expression of other non-HLA class I beta 2m-associated molecules. It was found that leukaemic B-cells at intermediate/late stages of differentiation, represented by non-Hodgkin's lymphoma (B-NHL) and 'hairy-cell' leukaemia (HCL), had significantly higher beta 2m:HLA-ABC ratios than did the cells from other types of B-cell malignancy. Although leukaemic B cells with a demonstrable non HLA-ABC-associated beta 2m component expressed detectable levels of CD1c, and insignificant levels of CD1a and CD1b, the antigen density was insufficient to account for the excess beta 2m. In vitro stimulation of leukaemic B cells by phorbol ester substantially increased the expression of HLA-ABC and CD1c, but also accentuated further the difference between the expression of these molecules and that of beta 2m. There was no detectable beta 2m other than that associated with HLA-ABC and CD1 on the surface of malignant T cells by contrast. Our findings strongly support the existence, at certain stages of leukaemic B-cell differentiation, of an additional beta 2m component(s) other than that associated with HLA-ABC and CD1.     

Gene rearrangements in T-cell lymphoblastic lymphoma

This study presents an examination of the Ig heavy chain (IgH) and T-cell receptor gamma (TCRγ) genes in a series of 39 CD3-positive T-cell acute lymphoblastic leukaemia (ALL) cases with and without co-expression of CD79a; 30/39 cases had a rearrangement of the TCRγ genes and two of these 30 cases also demonstrated an IgH rearrangement. No cases had solely an IgH rearrangement. The conclusion of the study is that lymphoblastic lymphoma cases that are positive for CD3 are of T-cell lineage, regardless of CD79a expression. Copyright © 1999 John Wiley & Sons, Ltd.     

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma

AIMS: Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes. METHODS AND RESULTS: For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes. CONCLUSIONS: These novel monoclonal antibodies may be of value in routine diagnostic practice.     

Killing of human leukaemia/lymphoma B cells by activated cytotoxic T lymphocytes in the presence of a bispecific monoclonal antibody (alpha CD3/alpha CD19).

Bispecific antibodies (BsAb) can be used to retarget T cells irrespective of their specificity to certain target cells inducing target cell lysis. We have tested the efficacy of the BsAb SHR-1, directed against the T cell antigen CD3 and the B cell antigen CD19 to induce (malignant) B cell kill by T cells as measured in a 51Cr-release assay. Two cytotoxic T cell clones (CTL), expressing TCR alpha beta or TCR gamma delta, were effective in killing CD19 expressing B cell lines at different stages of differentiation in the presence, but not in the absence, of the BsAb. CD19- target cells were not killed. Fresh CD19+ leukaemia/lymphoma cells were also efficiently killed by SHR-1 preincubated CTL clones. In addition, phytohaemagglutinin (PHA) or CD3-activated IL-2 expanded peripheral blood mononuclear cells (PBMC) of normal donors did so after 2 weeks of stimulation. A concentration of 100 ng/ml of the BsAb was sufficient to obtain optimal lysis of all target cells tested. These results show that fresh human leukaemia/lymphoma cells, freshly derived from active lymphoblastic leukaemia (ALL) as well as non-Hodgkin's lymphoma (NHL) patients, can be effectively killed in the presence of this BsAb by activated T cells.     

CD1c antigens are present in normal and neoplastic B-cells

The immunohistochemical detection of CD1c antigen is described in mantle zone B-cells of the tonsil, lymph node, and spleen, and also in the marginal zone B-cells of the spleen. CD1c expression was observed in most cases of low-grade, but in only a single case of high-grade, B-cell non-Hodgkin's lymphoma. It was not detected in germinal centre cells, nor in Epstein-Barr virus-transformed or Burkitt's lymphoma B-cell lines. This distribution suggests that CD1c expression may occur preferentially in slowly proliferating B-cell populations and does not support previous suggestions that CD1c is a human equivalent of the mouse thymus leukaemia antigens.     

Immunohistochemical and genetic analysis of Chinese nasal natural killer/T-cell lymphomas

Nasal natural killer/T-cell lymphoma (N-NK/T-L) is prevalent in China. To further characterize this neoplasm, 36 cases of N-NK/T-L from 304 cases of malignant lymphomas in the north China area were investigated by histopathology, immunophenotyping, and genomic analysis of c-kit, in comparison with 11 cases of B-cell lymphoma (BCL) at the same region and 5 cases of nodal peripheral T-cell lymphoma (PTCL) (unspecified). Histopathologically, N-NK/T-L was characterized by coagulative necrosis, inflammatory background, and angiodestructive growth pattern. In 36 cases of N-NK/T-L, 27 cases (75.0%) were stained for CD45RO and 25 (72.2%) for CD3epsilon. Thirty cases (83.3%) were positive for T-cell intracellular antigen-1, 22 (61.1%) for granzyme B, 18 (50.0%) for CD56, and 11 (30.6%) for CD30, whereas none was positive for CD117. All 5 cases of PTCL displayed positive staining for CD45RO and T-cell intracellular antigen-1, 3 cases for CD3epsilon, but only 1 case for granzyme B. All 11 BCLs presented positive staining for CD20 and CD79a but negative for other antibodies. A significant relationship was observed between neoplastic cells pleomorphism and granzyme B expression (P < .05). Despite the fact that all cases were negative for CD117 staining, genomic sequences of c-kit 11 and exon 17 sequencing showed that 8 (26%) of 31 cases N-NK/T-L proved to contain genomic mutations, including 4 cases in exon 11 and 4 in exon 17. For the control group, only 1 (9%) of 11 BCLs and 1 (20%) of 5 cases of PTCL were detected to harbor mutations in exon 11. All mutations detected in 3 groups were missense by base substitution, and codes 571, 572, and 821 were hot spots. The results suggested that, in addition to histological features and routine immunophenotyping, granzyme B expression should be a more reliable marker in correct diagnosis of N-NK/T-L, and genetic analysis of c-kit mutation should be helpful in the diagnosis of this tumor.     

CD79a is heterogeneously expressed in neoplastic and normal myeloid precursors and megakaryocytes in an antibody clone-dependent manner

CD79a, a component of the B-cell antigen receptor complex, can also be expressed in certain non-B-cell malignancies. The reported frequency of CD79a expression in acute myeloid leukemias (AML) ranges from 0% to 90%. We evaluated 39 bone marrow biopsy specimens (29 AML and 10 normal cases) using 5 different commercially available anti-CD79a monoclonal antibody (MoAb) clones. Of 7 acute promyelocytic leukemia (APL) cases, 6 (86%) stained for CD79a with clones HM47/A9 (Novocastra, Newcastle Upon Tyne, England) and HM57 (DAKO, Carpinteria, CA) but were negative with clones 11E3 (Novocastra), and JCB117 (DAKO). Half of 6 acute megakaryoblastic leukemia (AMKL) cases and normal megakaryocytes in 14 (67%) of 21 cases were immunoreactive using clone 11D10 (Novocastra). Approximately one third of non-APL/non-AMKL AML and myeloid precursors in normal marrow specimens stained with clones HM57 and 11D10. This heterogeneity of CD79a expression in AML, megakaryocytes, and myeloid precursors is MoAb clone-dependent, likely owing to different epitope detection, and may be of diagnostic usefulness.     

Peripheral T-cell lymphoma with aberrant expression of CD79a and CD20: a diagnostic pitfall.

Immunohistochemical studies are increasingly used for the routine diagnosis of lymphomas as it is widely accepted that lymphomas of different cell lineages vary in their prognosis and response to therapy. A case of peripheral T-cell lymphoma with aberrant expression of B-cell-associated antigens L-26 (CD20) and mb-1 (CD 79a) is described. The disease pursued an aggressive clinical course, and the patient died of disease 6 weeks after presentation. Immunohistochemical studies demonstrated expression of both T- and B-cell-associated antigens, including CD3, CD8, CD43, TIA-1, CD20, and CD79a. Other markers expressed by the tumor cells included CD56 and S-100. Of interest, betaF-1 staining for the beta chain of T-cell receptor (TCR) complex was positive in the small admixed T lymphocytes but was negative in the tumor cells, raising the possibility of a gamma/delta T-cell lymphoma. Molecular studies by polymerase chain reaction (PCR) demonstrated clonal TCR-gamma chain gene rearrangement without evidence for a clonal rearrangement of the immunoglobulin heavy chain gene. PCR for HHV-8 related sequences was negative. Mb-1 is an IgM-associated protein that was thought to be restricted to normal and neoplastic B cells. Although its coexpression has been reported in up to 10% cases of precursor T-cell lymphoblastic lymphoma, the coexpression of both CD20 and CD79a has not been described in mature T-cell malignancies. Biphenotypic lymphomas associated with HHV-8 have been reported in immunodeficiency, but no evidence of immune deficiency was identified, and studies for EBV and HHV-8 were negative. This case illustrates that no marker has absolute lineage specificity and that immunophenotypic studies should always be performed with panels of monoclonal antibodies. Moreover, cases with ambiguous phenotypes may require genotypic studies for precise lineage assignment.     

Precursor B lymphoblastic lymphoma presenting as lytic bone lesions

Precursor B lymphoblastic lymphoma is an aggressive but potentially curable disease. This lymphoma most often manifests in the skin and lymph nodes and, less commonly, as lytic bone lesions. In the bone, this lymphoma must be differentiated from small round blue cell tumors, diffuse large B-cell lymphoma, and acute myelogenous leukemia. We describe the morphologic and immunophenotypic features in 4 patients, 2 children, 1 teenager, and 1 adult, who initially presented with bone pain and osteolytic lesions but without peripheral blood or iliac bone marrow involvement. Positive immunohistochemical staining of the neoplastic cells was observed for anti-CD10 (3/4), CD20 (3/4), CD34 (1/4), CD43 (4/4), CD45/CD45RB (2/4), CD79a (4/4), CD99 (MIC2) (2/4), and terminal deoxynucleotidyl transferase (4/4). CD3 was absent in all cases. Immunophenotyping these neoplasms is essential to establish the correct diagnosis of precursor B lymphoblastic lymphoma, and a panel of antibodies is required because of the immunophenotypic heterogeneity.     

CD79a(+) T-cell lymphoblastic lymphoma with coexisting Langerhans cell histiocytosis.

Although there is a close association between Langerhans cell histiocytosis and malignant neoplasms, simultaneous occurrence of lymphoblastic lymphoma and Langerhans cell histiocytosis in the same lymph node is an extremely rare finding. Herein, we describe such a case in a 26-year-old woman who presented with progressive cervical lymphadenopathy. The lymphoma cells have an immature T-cell phenotype (terminal deoxynucleotidyl transferase(+), HLA-DR(+), CD34(+), CD38(+), and CD7(+)) with expression of both CD3 and CD79a on immunohistochemical stain. The Langerhans cells are present focally with the characteristic morphologic features and immunophenotype (CD1a(+) and S100(+)). The significance of CD79a coexpression in T-cell lymphoblastic lymphoma and the association between lymphoblastic lymphoma and Langerhans cell histiocytosis are discussed.     

Monocytes mediate shaving of B-cell-bound anti-CD20 antibodies

Anti-CD20 monoclonal antibodies are promising for the treatment of B-cell malignancies such as chronic lymphocytic leukaemia and autoimmune diseases where auto-antibodies play an important role. Anti-CD20 such as rituximab (RTX) mediates B-cell depletion through mechanisms such as complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. However, in haematological malignancies, such effector mechanisms can be saturated and result in release of malignant B cells with reduced levels of CD20. It has been hypothesized that this is the result of monocyte-mediated shaving of the CD20/RTX complex from the B-cell surface. Here, we confirm, that in vitro co-culture of human monocytes and RTX-labelled syngeneic B cells results in reduced expression of CD20/RTX complex on the B cell surface. This shaving mechanism was the result of active protease activity because EDTA and PMSF were able to mediate partial inhibition. Also, a series of alternative anti-CD20 antibodies representing both type I and type II antibodies were tested for their ability to induce the shaving reaction. These results demonstrate that a monocyte-mediated shaving reaction can lead to complete loss of most anti-CD20 antibodies from the surface of B cells even from healthy donors and this is an important obstacle for antibody-mediated immune therapy. The findings demonstrate the necessity of developing novel antibodies that maintain high effector functions without enabling activation of the shaving reaction.     

Monoclonal antibody L26: an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin wax embedded tissues.

Formalin fixed and paraffin wax embedded tissue from 85 well characterised cases of non-Hodgkin's lymphoma and Hodgkin's disease were studied using the avidin-biotin-peroxidase complex technique. Among the non-Hodgkin's lymphomas all cases of B cell lymphoma were reactive with L26, a monoclonal antibody which is as yet an unclustered pan B cell reagent, with the exception of pre-B cell acute lymphoblastic leukaemia and malignant lymphoma plasmacytic. Eighteen well characterised cases of T cell lymphoma, selected to include tumours previously shown to exhibit cross reactivity with antibodies to fixation resistant B cell related antigens, were similarly studied. Neoplastic cells in all but one case were unstained by L26. Twenty seven cases of Hodgkin's disease were also examined. In five cases all Reed-Sternberg cells and their variants were strongly stained by L26; only a proportion of Reed-Sternberg cells and their variants were recognised in a further five cases. Monoclonal antibody L26 promises to be a valuable reagent for the diagnosis of malignant lymphoma in routinely fixed and paraffin wax embedded tissues. Its advantage lies in its sensitivity and greater B cell specificity than any of the B cell related reagents currently available for the study of malignant lymphoma in fixed tissues.     

Heterogeneity of CD44 expression among human B-cell subpopulations

Abstract: CD44 is a widely distributed cell surface glycoprotein that participates in a number of cellular adhesion and signal transduction processes. Germinal center B cells express very low levels of CD44, whereas their precursors and differentiation products express much higher levels. In immunofluorescence studies comparing 20 antibodies classified as being against the hematopoietic isoform of CD44, one antibody, AIG3, was unreactive with germinal center B cells, whereas the other antibodies showed low intensity but definite reactivity. Western blotting and sequential immunoprecipitation studies of lysates from density-separated lymphocyte fractions showed two bands that were differentially expressed and reacted differently with AIG3 compared with the other CD44 antibodies. These results suggest that germinal center B cells and non-germinal center B cells express different forms of CD44.Of 21 malignant B-cell populations examined, 5 showed reactivity with a "standard" CD44 reagent and significantly reduced reactivity with AIG3, while one sample showed the opposite pattern and the remainder were positive for both reagents. Of 10 cell lines studied, 5 were differentially stained by AIG3 and a standard CD44 antibody. PCR amplification of reverse transcribed mRNA from sorted human tonsil B-cell subpopulations and Southern blotting showed that B cells express a number of splice isoforms of CD44. These results demonstrate that B cells express multiple forms of CD44; both splice insert isoforms and variants distinguished by AIG3; the form of CD44 expressed depends on B-cell differentiation state.