Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study.     

Expression of lymphocyte homing receptors and vascular addressins in low-grade gastric B-cell lymphomas of mucosa-associated lymphoid tissue.

In this study, we examined lymphocyte homing receptor and vascular addressin expression in a case of primary gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) with a secondary intestinal spread. We compared the findings with that observed in B cells of normal MALT and MALT acquired as a consequence of Helicobacter pylori-associated gastritis and other low-grade gastric B-cell MALT lymphomas. The neoplastic B cells in the gastric tumor were alpha 4 beta 7-, CD62L+, whereas the intestinal secondary was alpha 4 beta 7+, CD62L-. Incubation of isolated tumor cells from the stomach by H. pylori generated T-cell-dependent proliferation of neoplastic B cells and induced expression of alpha 4 beta 7 integrin similar to the intestinal tumor. These observations indicate that reversal of homing receptor profile in the gastric tumor by antigen specific stimulation may be responsible for secondary intestinal dissemination. In normal stomach and normal MALT, alpha 4 beta 7 and CD62L expression reflected the differentiation of the B cell. Plasma cells were alpha 4 beta 7+, CD62L-, whereas a subset of memory B cells were alpha 4 beta 7-, CD62L+. Homing receptor expression in MALT lymphoma B cells was heterogeneous, however, in line with their memory B-cell phenotype in the majority of cases, the neoplastic B cells were alpha 4 beta 7-, CD62L+. Neoplastic plasma cells were always alpha 4 beta 7+, CD62L-. The venules in normal gastric mucosa expressed mucosal addressin cell adhesion molecule-1 but not peripheral lymph node addressin. In normal MALT, H. pylori-associated follicular gastritis and MALT lymphomas high endothelial venules coexpressed mucosal addressin cell adhesion molecule-1 and peripheral lymph node addressin. These findings suggest expression of lymphocyte homing receptors by B cells and vascular addressins by mucosal venules are similar in normal MALT and MALT lymphomas, and factors controlling normal mucosal B-cell traffic are also operational in MALT lymphomas.     

Immunocytochemical analysis of MNDA in tissue sections and sorted normal bone marrow cells documents expression only in maturing normal and neoplastic myelomonocytic cells and a subset of normal and neoplastic B lymphocytes.

The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear antigen known to be expressed in mature myelomonocytic cell lines. An extensive immunocytochemical evaluation of fixed tissues confirmed MNDA expression in normal maturing granulocytes and monocytes and in acute nonlymphocytic leukemias and chronic myelogenous leukemia. MNDA was not detected in normal tissue histiocytes but was found in activated macrophages and foreign body giant cells associated with inflammation. Flow cytometric cell sorting of normal bone marrow established that MNDA is initially expressed in myeloid blast cells. Examination of lymphoid tissues showed a low level of expression in a population of normal mande B lymphocytes but not in germinal center cells or plasma cells. A subset of B cell neoplasms expressing MNDA included hairy cell leukemia, parafollicular (monocytoid) B cell lymphoma, mantle cell lymphoma, and small lymphocytic lymphoma. Cell sorting of normal bone marrow showed MNDA expression in CD20+/CD10-/CD5- B cells. MNDA was not detected in other normal bone marrow or all other nonhematopoietic cells. The hematopoietic cell-specific pattern of MNDA expression was elucidated through a comprehensive analysis of normal and neoplastic tissues, and the results provide further evidence of the coexpression of B- and myeloid cell markers in neoplastic B cells and identify a normal B cell population that might be related to the cell of origin of a subset of B cell neoplasms.     

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.     

Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies.

Two distinct antigen systems (L26 and L27) specifically expressed in human B lymphocytes were identified using TB2-2B3 (2B3) and T3-5B3 (5B3) monoclonal antibodies, respectively. Whereas L26 antigen defined by 2B3 were rarely expressed on the surface of B cells but abundant in the cytoplasm, 127 antigens detected by 5B3 was clearly expressed on the cell surface. These two antigens appeared to be restricted in their expression to B cells, as they were found in most B cells but not other cell types including thymocytes, T cells, monocytes and granulocytes. Functional studies demonstrated that L27 was more easily lost from B cells after activation with pokeweed mitogen than was L26. Likewise, plasma cell myeloma, as well as normal plasma cells, was devoid of both L26 and L27, whereas immunoblastic sarcoma of B cell type expressed L26 but not L27. These two antigens co-existed in the same B cell lines including Epstein-Barr virus transformed B cell lines, B cell type acute lymphatic leukaemia (B-ALL) cell line, Burkitt's lymphoma cell lines and myeloma cell lines, but pre-B and common ALL cell lines were entirely negative for both L26 and L27. Immunoprecipitation studies showed that L26 consisted of at least two polypeptide chains with molecular weights of 30K and 33K daltons, which were clearly distinct from HLA-DR antigens. The antigen L27 is presently under study.     

Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody

The novel gene H731 (approved name: PDCD4 (programmed cell death 4)) has been isolated as an antigen gene of the monoclonal antibody Pr-28 which recognized a nuclear antigen in proliferating cells. The gene is homologous to the mouse gene (MA-3/Pdcd4/A7-1) which was associated with apoptosis and was shown to suppress tumor promoter-induced neoplastic transformation. A polyclonal antibody against H731-protein derived from an extract of Escherichia coli transformed with an H731 expression plasmid was prepared, and the H731-protein expression in human normal and tumor cells using the antibody was studied. The staining patterns of asynchronous cultures of human normal fibroblasts (MRC-5) were heterogeneous but the antigen was accumulated in the nuclei at the G0 phase. On the contrary, the antigen was overproduced and localized in the cytoplasm during the cell cycle in tumor cell lines. Immunohistological studies revealed that the H731-protein was highly expressed in bladder carcinoma and breast carcinoma tissues compared with the normal tissues so far tested. These results indicated that expression of the H731-protein was up-regulated or induced in the proliferative cells. Immunohistological studies also revealed that the protein was abundantly expressed in the small duct epithelial cells of the normal mammary gland.     

Utilization of monoclonal antibody L26 in the identification and confirmation of B-cell lymphomas. A sensitive and specific marker applicable to formalin-and B5-fixed, paraffin-embedded tissues.

Immunophenotypic analysis of paraffin-embedded tissues of lymphoproliferative disorders has been facilitated by recent developments of monoclonal antibodies that react with epitopes that survive histologic processing. Leukocyte common antigen (LCA) antibody has made a significant contribution to the immunocytochemical separation of non-Hodgkin's lymphomas from nonlymphoid neoplasms. However, a small percentage of lymphomas, particularly some large cell or immunoblastic B-cell tumors, will not label with LCA antibody. Other antibodies, directed against B lymphocytes, experience problems of specificity and a lack of sensitivity when applied to formalin-fixed specimens. The authors recently investigated a monoclonal antibody (L26) that demonstrates excellent specificity and sensitivity for B lymphocytes, and tumors derived from them, in formalin- and B5-fixed, paraffin-embedded tissue. The avidin-biotin peroxidase complex (ABC) technique was utilized for immunostaining 95 cases of malignant lymphoproliferative disorders and a variety of normal and neoplastic nonlymphoid tissues. When applied to sections of benign lymphoid tissue, the L26 antibody labeled germinal center cells, mantle zone and scattered interfollicular lymphocytes, but not histiocytes or plasma cells. L26 marked 100% (44/44) of the large cell and immunoblastic B-cell lymphomas, along with 1 case of pre-B cell lymphoblastic lymphoma. This included 8 cases that were LCA-negative. None of the T-cell lymphomas or plasma cell tumors studied demonstrated L26 immunostaining. No normal, benign, or neoplastic nonlymphoid tissues examined stained with this antibody. L26 successfully labels B lymphocytes and B-cell lymphomas in routinely processed tissues, often with greater sensitivity and intensity than LCA. This antibody should prove invaluable in the investigation of atypical lymphoid proliferations and the identification of B-cell derived lymphomas, when fresh or frozen tissue is unavailable for analysis.     

A novel monoclonal antibody (OPD4) recognizing a helper/inducer T cell subset. Its application to paraffin-embedded tissues.

A novel monoclonal antibody (MAb), OPD4, reactive with a helper/inducer (H/I) subset of T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with an activated H/I T cell line, namely DL40. The antibody is an IgG1 antibody and it recognizes an antigen with a molecular weight of 200 kd, corresponding to that of leukocyte common antigen. OPD4+/CD4+ T cells provided better help for pokeweed mitogen-stimulated polyclonal IgG production than OPD4-/CD4+ T cells. OPD4 recognized the H/I T cell subset even in paraffin-embedded tissue sections, but did not recognize nonhematopoietic cells, suppressor/cytotoxic T cells, B cells, monocytes in the peripheral blood, or other normal hematopoietic cells as examined by the flow cytometric and immunoperoxidase methods. Besides the lymphoid cells, OPD4 reacted with a number of histiocytes (epithelioid cells) in tissues from sarcoidosis and tuberculosis. For the neoplastic lesions, OPD4 reacted with approximately half of the cases of T cell lymphomas. Consequently, OPD4 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.     

Expression of CD31 epitopes on human lymphocytes: CD31 monoclonal antibodies differentiate between naive (CD45RA+) and memory (CD45RA-) CD4-positive T cells

The CD31 antigen, a member of the immunoglobulin superfamily with a possible cell adhesion function, is expressed on approximately 50% of peripheral blood lymphoid cells at relatively low intensity (10-20% of the level on monocytes). In the accompanying paper we showed that a mAb, 5A2.G5, which identifies a glycosylation-dependent epitope of the CD31 antigen, bound to fewer lymphocytes than two other CD31 mAb, B2B1 and 2BD4, although the 3 antibodies bound equally well to monocytes. We have now analyzed the pattern of expression of epitopes of the CD31 antigen on lymphoid cell subpopulations using two-color immunofluorescence and flow cytometry. Large granular lymphocytes (CD16+), CD8-positive T cells and B cells (SMIg+) were mostly CD31-positive as indicated by the binding of mAb B2B1 and 2BD4. Single populations displaying some overlap with the negative control were obtained in each case. In contrast, CD4-positive T cells fell into two discrete populations with respect to CD31 antigen expression. mAb 5A2.G5 displayed weaker binding to all lymphoid cell types, indicating that the pattern of glycosylation of the CD31 antigen differs between lymphocytes (of all types) and cells of the myeloid lineages. The heterogeneity of CD31 antigen expression by CD4-positive cells was further examined by dual-labelling of purified CD4 cells with mAb B2B1 and CD45RA or CD29 mAb which identify naive and memory T cells respectively. The CD31 antigen was found to be preferentially expressed by the CD45RA-positive, naive cell population.     

Podoplanin (d2-40): a new immunohistochemical marker for reactive follicular dendritic cells and follicular dendritic cell sarcomas

The diagnosis of follicular dendritic cell (FDC) sarcoma can be challenging because of its morphologic overlaps with many other spindle cell neoplasms and, therefore, new phenotypic markers will be helpful in its differential diagnosis. Podoplanin is a mucin-type transmembrane glycoprotein that has recently been detected in reactive FDCs. In this study, we investigated the expression patterns of podoplanin using a new mouse monoclonal antibody D2-40, and compared them with CD21, a well-established FDC marker, in a comprehensive panel of cases. The panel included 4 FDC sarcomas, 38 spindle cell neoplasms of other types, 25 reactive lymphoid hyperplasia, and 117 lymphoid and 5 myeloid malignant hematopoietic neoplasms. Our study revealed that D2-40 strongly stained 3 of 4 FDC sarcomas. In contrast, D2-40 stained only 2/38 other spindle cell neoplasms tested. Furthermore, we observed that D2-40 highlighted more FDC meshworks than CD21 in Castleman's disease, follicular lymphoma, nodular lymphocyte predominance Hodgkin lymphoma, and residual reactive germinal centers in a variety of lymphoma types. D2-40 and CD21 stained an equal number of cases of reactive lymphoid hyperplasia, progressively transformed germinal centers and angioimmunoblastic T-cell lymphoma. No expression of podoplanin was detected in normal or neoplastic lymphoid and myeloid cells. We conclude that podoplanin (D2-40) is a sensitive and specific FDC marker, which is superior or equal to CD21 in evaluating both reactive and neoplastic FDCs. In addition, our results suggest that podoplanin (D2-40) can be used to support the diagnosis of FDC sarcoma.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

分离培养人外周血CD4+CD25-T细胞及其生物学特性研究

目的:分离培养人外周血CD4 CD25-T细胞,并鉴定其生物学特性。方法:设对照组(A组)、LPS组(B组)、LPS 抗TGF-β1mAb组(D),用Percoll不连续密度梯度离心与免疫磁珠法,分离培养健康人外周血CD4 CD25-T细胞。用光镜及电镜观察其形态特征,台盼蓝试验检测其活力,流式细胞术(FCM)鉴定其纯度。体外培养4h、3d及5d后,用FCM检测CD4 CD25 T细胞的阳性率,ELISA法检测培养上清中TGF-β1的浓度,RT-PCR法检测细胞中叉状头/翼状螺旋转录因子(FOXP3)mRNA的表达。结果:(1)光镜下观察分离的CD4 CD25-T细胞主要为小体积细胞,电镜下观察细胞核呈圆形,染色质致密。体外抗人CD3/CD28mAb刺激培养的CD4 CD25-T细胞体积逐渐增大,胞质较丰富,电镜下观察细胞核呈椭圆形或肾型,染色质较稀疏。(2)FCM检测CD4 CD25-T细胞纯度达91.5%～96%。台盼蓝试验检测分离前后活细胞数无统计学意义(P>0.05)。(3)FCM检测表明,B5d组为CD4 CD25 T细胞的阳性率为(55.99±1.42)%与A5d组相比较有统计学意义(P<0.01);D5d组CD4 CD25 T细胞的阳性率为(1.99±0.83)%与A5d组的阳性率(1.29±0.04)%相比较无统计学意义。(4)ELISA测定表明,培养液中TGF-β1的浓度,B3d组为(1.60±0.09)μg/L、B5d组为(1.83±0.14)μg/L,分别与A3d组为(0.35±0.04)μg/L、A5d组为(0.33±0.08)μg/L相比较,均有统计学意义(P<0.01);而D3d、D5d组与A3d、A5d组相比较均无统计学意义。(5)RT-PCR检测表明,FOXP3mRNA的表达:以β-actin的A值作为内参照,B3d组为(0.84±0.07)、B5d组为(1.85±0.24)分别与A3d组(0.05±0.02)、A5d组(0.04±0.02)相比较,均有统计学意义(P<0.01);而D组与A组的各个组间相比较均无统计学意义。(6)LPS诱导的人外周血中CD4 CD25-T细胞培养液上清中TGF-β1的水平与该细胞中FOXP3mRNA的表达呈显著的正相关(r=0.812,P<0.01)。结论:用Percoll不连续密度梯度离心与免疫磁珠法体外分离培养的人外周血CD4 CD25-T细胞的活力及纯度较理想;LPS诱导的CD4 CD25-T细胞中FOXP3mRNA的表达,可能与TGF-β1有关。     

Isolation and EBV-transformation of a minor population of normal human tonsillar B cells bearing a cold agglutinin-associated idiotope

Human autoantibodies which bind to red blood cells and cause agglutination in the cold, collectively comprise the cold agglutinins: a subset of these antibodies with specificity for the Ii carbohydrate antigen on the red cell surface bears cross-reacting idiotypic determinants. A monoclonal antibody has been raised which specifically inhibits the cold agglutination reaction, and has been used to probe normal lymphoid cell populations for the presence of B cells expressing the same idiotope. Although such cells comprise only 1-2% of the lymphoid cells in the tonsil, it has been possible to isolate them in good yield and purity using the antibody and magnetic beads. Released cells were infected with the Epstein-Barr (EB) virus and an idiotope-bearing line established. The IgM secreted by the line was found to agglutinate red cells in the cold indicating that cold agglutinin-producing cells were among this idiotope-positive population. The immunophenotype of the line, and the agglutinating power of the secreted IgM, have been compared with a similarly immortalized idiotope-bearing line established from neoplastic B cells of a patient with frank cold agglutination disease.     

Hairy cell leukemia-associated antigen LeuM5 (CD11c) is preferentially expressed by benign activated and neoplastic CD8 T cells.

LeuM5 antigen (CD11c, p150.95) expression, widely used as an immunodiagnostic marker for B-cell hairy cell leukemia, was examined on benign, normal peripheral blood T cells before and after stimulation in vitro with phytohemagglutinin and on a large, diverse panel of 73 T-cell neoplasms. Resting T cells lacked LeuM5. Intracytoplasmic LeuM5 was detectable at 3 to 4 days and surface membrane LeuM5 was detectable continuously between 5 and 17 days on greater than or equal to 20% CD3 cells (maximum, 42% CD3 cells at 10 days) after activation. Two-color flow cytometric analysis of the activated T cells demonstrated that a maximum of 60% CD8 but only 25% CD4 cells expressed LeuM5; the mean percentage of LeuM5+ CD8 cells was 44% compared with 12% LeuM5+ CD4 cells. A variable proportion of the neoplastic T cells in 19 of 73 (26%) T-cell neoplasms were LeuM5+. Twelve of 18 CD4-CD8+ (67%) but only 5 of 40 CD4+ CD8- T-cell neoplasms expressed LeuM5. These studies demonstrate that the LeuM5 antigen is 1) expressed in association with T-cell activation, 2) preferentially expressed by activated CD8 cells, and 3) variably expressed by neoplastic T cells, but particularly by those exhibiting the CD4- CD8+ phenotype.     

A novel pathway of human B cell activation initiated by CK226 surface antigen

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75-kDa surface molecule expressed on both T and B lymphocytes and to mediate T lymphocyte activation and proliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40-80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up-regulation of major histocompatibility complex class II and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either T cells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12-myristate 13-acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226-induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti-CD25 mAb. Addition of exogenous interleukin 2 to CK226-stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co-stimulatory effect with submitogenic concentrations of either anti-Ig antibody or Staphylococcus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose-dependent fashion by CK226 mAb.     

Monoclonal antibody Leu-22 (L60) permits the demonstration of some neoplastic T cells in routinely fixed and paraffin-embedded tissue sections

Monoclonal antibody Leu-22 (L60) detects a T cell-associated antigen which is stably expressed in routinely fixed and paraffin-embedded tissue sections. We investigated the utility of monoclonal antibody Leu-22 to immunophenotype routinely processed lymphoid neoplasms by determining its reactivity in 105 archival pathologic specimens of lymphoid neoplasia that had been previously immunophenotyped by standard cell suspension and frozen tissue section techniques. Monoclonal antibody Leu-22 reacted with 69% of T cell non-Hodgkin's lymphomas (NHLs), including cases belonging to each of the major clinicopathologic categories, and with 22% of B cell NHLs, but did not react with the Reed-Sternberg (RS) cells of Hodgkin's disease (HD). We concluded that monoclonal antibody Leu-22 reacts preferentially but not exclusively with T cell NHLs. Therefore, we performed parallel analyses of the same 105 cases with monoclonal antibodies leukocyte common antigen (LCA), Leu-M1, LN1, and LN2, which detect various paraffin-resistant antigens, and of 80 of these cases with monoclonal antibody UCHL1, which detects a paraffin-resistant T cell-associated antigen. UCHL1 reacted with 61% of the T cell NHLs studied. Sixty-nine percent of T cell NHLs expressed the LCA+, Leu-22+ or Leu-M1+, LN1- phenotype and 47% of B cell NHLs expressed the LCA+, Leu-22-, Leu-M1-, LN1+ phenotype. These phenotypes had a false-positive rate of only 7%. The substitution of UCHL1 for Leu-22 or the combined use of UCHL1 and Leu-22 in this panel did not improve our ability to correctly predict the T cell phenotype of these lymphoid neoplasms. LN1 and LN2 reacted with 13% and 56% of T cell NHLs, respectively, and LN2 reacted with RS cells in 85% of cases of HD. In summary, our results demonstrate that the judicious use of monoclonal antibody Leu-22 in combination with other selected commercially available monoclonal antibodies permits the determination of the B cell or T cell origin of a high proportion of NHLs, and is helpful in the differential diagnosis between HD and NHL among cases that have been routinely fixed and paraffin-embedded.     

Arachidonate 5-lipoxygenase establishes adaptive humoral immunity by controlling primary B cells and their cognate T-cell help

In this study, we report the unique role of arachidonate 5-lipoxygenase (Alox5) in the regulation of specific humoral immune responses. We previously reported an L22 monoclonal antibody with which human primary resting B cells in the mantle zones of lymphoid follicles are well-defined. Proteomics analyses enabled identification of an L22 antigen as Alox5, which was highly expressed by naive and memory B cells surrounding germinal centers. Cellular growth of mantle cell lymphoma cells also seemed to depend on Alox5. Alox5(-/-) mice exhibited weak antibody responses specific to foreign antigens at the initial and recall phases. This was probably attributable to the low number of follicular and memory B cells and the functional loss of interleukin-21-mediated responses of follicular B cells. Moreover, Alox5(-/-) mice could not fully foster the development of follicular B helper T (Tfh) cells even after immunization with foreign antigens. Further experiments indicated that Alox5 affected mortality in experimentally induced enterocolitis in germ-prone circumstances, indicating that Alox5 would endow immunologic milieu. Our results illustrate the novel role of Alox5 in adaptive humoral immunity by managing primary B cells and Tfh cells in vivo.     

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39

A human monoclonal IgM antibody, referred to as TU223, has been produced. The reactivity of TU223 was tested in various cells and cell lines by complement-dependent microcytotoxicity test and fluorescence-activated cell sorter analysis. The antigen defined by TU223 was expressed on Epstein-Barr virus-transformed B-cell lines and on some Burkitt's lymphoma cell lines, but was not expressed on normal T cells, B cells or erythrocytes. In addition, expression of the antigen defined by TU223 was also induced on B cells activated by Epstein-Barr virus or pokeweed mitogen, and on T cells activated by phytohemagglutinin, concanavalin A, pokeweed mitogen or recombinant interleukin-2. However, no expression of the antigen detected by TU223 was induced at all on recombinant interleukin-4-treated B cells or macrophage-like cell line U937. When the ability of TU223 and various mouse monoclonal antibodies to bind to human differentiation antigens was compared, interestingly, the reactivity of TU223 was found to be very similar to that of mouse monoclonal antibody CD23 (H107), which reacts with Fc epsilon receptor II. Two-color analysis revealed that the antigen defined by TU223 is expressed on the cell surface of certain lymphoid cells expressing CD23 antigen. However, it can be concluded that the antigen defined by TU223 is clearly distinct from Fc epsilon receptor II, based on assay of cross-blocking between H107 and TU223. The surface antigen on B85 cells recognized by TU2232 had the molecular size of 80-82 kiloDaltons as determined by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A unique human B lymphocyte antigen defined by a monoclonal antibody

We produced a hybridoma designated 4G7 from a mouse immunized with chronic lymphocytic leukemia cells. The 4G7 hybridoma secretes an IgG1 antibody that is specific for normal and malignant B lymphocytes. Using dual color immunofluorescence staining, this antibody reacted with all immunoglobulin-positive cells but no T cells in normal peripheral blood. There was no detectable 4G7 antigen on monocytes, platelets, red cells, granulocytes, or phytohemagglutinin-activated T cells. When PBL were depleted of 4G7 positive cells and stimulated with pokeweed mitogen, secreted immunoglobulin levels fell to less than 10% of control values on Day 5 and less than 1% of control on Day 7. This antibody was reactive with 155 of 176 B lineage neoplasms on which it was screened. Thirty-five cases of myeloid or T-lymphoid malignancy were negative. Our studies show that the 4G7 antigen modulates in the presence of excess antibody. Free 4G7 antigen was not found circulating in human serum. The cell surface antigen identified by 4G7 was sensitive to pronase proteolysis but resistant to trypsin and chymotrypsin digestion. A comparison of 4G7 with other known B-cell antibodies indicates that the 4G7 antigen has not been previously identified. This antibody is of use for the identification of normal B lymphocytes, the study of B-cell differentiation, and the characterization of lymphoid malignancies.     

A human T cell tumor which expresses the putative T cell antigen receptor

A monoclonal antibody (mAb) called H1-2D4 which reacts only with the T cell line HPB-ALL and not with other T cell lines, normal or activated peripheral blood cells, B lymphoblasts (B-LCL), or thymocytes has been developed. This mAb cocaps the T3 antigen on HPB-ALL and anti-T3 mAb cocaps the antigen which reacts with H1-2D4. Purified H1-2D4 precipitates a heterodimer from HPB-ALL cells which has components with molecular weights of 51000 and 39000. The properties of the molecule recognized by H1-2D4 suggest that it is the HPB-ALL equivalent of the putative human T cell antigen receptor.