Advance of Research on the Immunotherapy Targeting B Cell Maration Antigen for Multiple Myeloma--Review / 中国实验血液学杂志

Abstract　　B cell maturation antigen (BCMA) is an ideal target for precise treatment due to its highly selective expression on malignant myeloma cells. This review summarizes briefly the advances in the latest research progress on biological activity of BCMA, its significance as a biomarker and immunotherapy direcited against BCMA, such as bispecific antibodies, antibody drug conjugates, chimeric antigen receptor T cell therapy against mature B cell antigens.

Relevancia pronóstica de la proteína B cell lymphoma 2 (bcl-2) en los subtipos moleculares intrínsicos del carcinoma de mama / Prognostic relevance of the B cell lymphoma 2 protein (bcl-2) in the intrinsic molecular subtypes of breast carcinoma

Introducción: El B cell lymphoma 2 (Bcl-2) es una proteína anti-apoptótica que promueve la supervivencia de las células tumorales. A pesar de lo anterior, en el cáncer de mama se ha informado que su expresión es un factor pronóstico favorable. Objetivo: Evaluar el valor pronóstico de Bcl-2 según el subtipo molecular en mujeres venezolanas con cáncer de mama. Métodos: La relación entre la expresión inmunohistoquímica (IHQ) de Bcl-2, las variables clínico-patológicas y la supervivencia global (SG) media se analizó en 178 pacientes, cuyos tumores se clasificaron de acuerdo a lo establecido en el Consenso de St. Gallen 2015 y en función de la expresión IHQ de los receptores hormonales (estrógeno y progesterona), el receptor del factor de crecimiento epidérmico humano 2 (HER2) y el índice de proliferación (Ki-67): Luminal A, Luminal B, HER2 y Triple Negativo (TN). Resultados: Un total de 84 pacientes (47 %) tuvieron tumores positivos para Bcl-2, los cuales se asociaron significativamente con un grado histológico bajo (I y II), estadio clínico iniciales (I y II) y Ki-67 (<20 %). En los subtipos Luminales la expresión de Bcl-2 mostró un pronóstico mejor pero sin significancia estadística. Por el contrario, hubo un efecto pronóstico significativo (P=0,017) de la expresión de Bcl-2 en el subtipo TN, con SG media significativamente menor en comparación de la SG obtenida en los tumores Luminal A con Bcl-2 positivo. Conclusión: La expresión de Bcl-2 es un marcador de buen pronóstico en todos los subtipos moleculares, con significancia estadística sólo en el subtipo TN (AU)

Smoking promotes exacerbated inflammatory features in dendritic cells of Chilean rheumatoid arthritis patients / El tabaco exacerba características inflamatorias en células dendríticas de pacientes chilenos con artritis reumatoide

ABSTRACT Background: The dual potential to promote tolerance or inflammation when facing self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. There is an association between smoking and DCs maturation in patients with rheumatoid arthritis (RA). However, ethnicity is a key factor in autoimmune disorders. Aim: To evaluate phenotypic and functional alterations of DCs obtained from Chilean patients with RA as compared to healthy controls (HC). In second term, to compare the inflammatory behaviour of DCs between smoker and non-smoker patients. Material and Methods: Monocyte-derived DCs and T-cells were obtained from blood samples isolated from 30 HC and 32 RA-patients, 14 of which were currently smokers and 18 non-smokers. Several maturation surface markers were evaluated in DCs, including HLA-DR, CD40, CD80, CD83 and CD86. Furthermore, autologous co-cultures of DCs and T-cells were carried out and then T-cell proliferation, and expansion of Th1, Th17 and Tregs were analysed. Results: Compared with HC, RA-patients displayed increased HLA-DR expression in DCs, which was manifested mainly in patients with moderate-to- high disease activity scores (DAS28). Furthermore, RA-patients presented a stronger Th17-expansion and a correlation between DAS28 and Th1-expansion. Both effects were mainly observed in patients in remission or with a low DAS28. Moreover, smoker RA-patients displayed enhanced HLA-DR and CD83 expression in DCs as well as an exacerbated Th17-expansion and a correlation between DAS28 and Th1-expansion. Conclusions: These findings suggest that smoking enhances the inflammatory behaviour of DCs and the consequent Th1 and Th17-mediated response in patients with RA

Introducción: El potencial dual que poseen para promover tolerancia o inflamación ante antígenos propios, hace de las células dendríticas (CDs) actores fundamentales en el desarrollo de autoinmunidad. Existe una asociación entre fumar y la maduración de las CDs en pacientes con artritis reumatoide (AR). No obstante, la etnicidad es un factor clave a considerar en desórdenes autoinmunes. Objetivos: Comparar las alteraciones fenotípicas y funcionales de las CDs obtenidas desde pacientes Chilenos con AR y controles sanos (CS). Además, analizamos las diferencias en el comportamiento inflamatorio que existe entre las CDs obtenidas de pacientes fumadores y CS. Materiales y Métodos: Se obtuvieron CDs derivadas de monocitos y células T desde muestras de sangre aisladas de 30 CS y 32 pacientes con AR, 14 de los cuales eran fumadores y 18 no fumadores. Se evaluaron marcadores de maduración en la superficie de las CDs: HLA-DR, CD40, CD80, CD83 y CD86. Además, se realizaron co-cultivos autólogos de células T y CDs, analizando la proliferación de células T, y la expansión de células Th1, Th17 y Tregs. Resultados: En comparación con los CS, los pacientes AR mostraron un aumento de la expresión de HLA-DR en las CDs, principalmente en los individuos con DAS28 moderado-alto. Los pacientes con AR presentaron una mayor expansión de células Th17 y una correlación entre el DAS28 y la expansión de células Th1, ambos efectos manifestados principalmente en los individuos con un DAS28 bajo o en remisión. Además, los pacientes con AR fumadores mostraron un aumento en la expresión de HLA-DR y CD83 en las CDs y una expansión de células Th17 exacerbada así como una correlación entre el DAS28 y la expansión de células Th1. Conclusiones: Nuestros resultados sugieren que fumar favorece el comportamiento inflamatorio de las CDs y en consecuencia la inducción de respuestas mediadas por células Th1 y Th17 en los pacientes Chilenos con AR.

CD74 expression in colorectal carcinoma, correlation with prognostic markers

This work aimed to study the expression of CD74, a protein which has an important function in antigen presentation by immune competent cells, in colorectal carcinoma to explore the feasibility of using it in antigen directed therapy in this type of cancer, we also aim to assess its prognostic value through studying its correlation with tumor grade, Ki 67 expression, and CD31 expression. Twenty three retrospective, formalin fixed, paraffin embedded specimens of colorectal carcinomas were graded using H and E stained sections after Bosman et al, [2010]. Specimens were stained using immunoperoxidase technique for CD74, Ki67, and CD31 antibodies, followed by semiquantitation of their immunoreactivities. The results were tabulated and statistically analysed using ANOVA test and Pearson's correlation coefficient. CD74 protein expression was present in 11/14 [79%] of adenocarcinoma NOS, with diffuse pattern of staining in all the positive cases except one case. The average weighted score [AWS] was 3.1: 4.5. CD74 was totally negative in mucin producing carcinomas [7cases]. CD74 was diffusely expressed in the studied case of neuroendocrine carcinoma but not in the case of medullary carcinoma. The correlation between CD74 and tumor grade was insignificant [p<0.2]. The average Ki-67 labeling index in colorectal carcinomas was 39%. The mean value for CD31 expression was 8.7: 14. Cd74 was positively correlated to Ki67 proliferation index [p<0.0004], as well as to CD31 expression [p<0.059]. Analysis of variance [ANOVA] for the three biomarkers [CD74, Cd31. Ki76] showed highly significant P value [p<0.000002]. In colorectal carcinoma CD74 expression positively correlates with Ki67 proliferation index and angiogenesis which suggests that it may be a marker of poor prognosis in such carcinoma. CD74 expressed in most of colorectal carcinomas suggesting the feasibility of using it in antigen directed immunotherapy in this type of cancer

ROS kinase fusions are not common in Chinese patients with cholangiocarcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expressions of different forms of ROS fusions in Chinese patients with cholangiocarcinoma (CCA).</p><p><b>METHODS</b>RT-PCR was employed to examine formalin-fixed and paraffin-embedded CCA samples from stage I-IV patients for detection of ROS fusions involving Fused in Glioblastoma (FIG), solute carrier protein (SLC34A2) and major histocompatibility complex class II invariant chain (CD74). Serpin peptidase inhibitor clade A member 1 (SERPINA1) was detected as the reference gene.</p><p><b>RESULTS</b>In all the 56 CCA samples, 80.4% (45/56) were positive for SERPINA1 expression as evaluable samples. Of these evaluable samples, none expressed the ROS fusions.</p><p><b>CONCLUSION</b>ROS fusions are not common in Chinese CCA patients.</p>

Silencing invariant chains of dendritic cells enhances anti-tumor immunity using small-interfering RNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Genetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed.</p><p><b>METHODS</b>The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used.</p><p><b>RESULTS</b>The Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05).</p><p><b>CONCLUSION</b>Silencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.</p>

Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.</p><p><b>METHODS</b>By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.</p><p><b>RESULTS</b>The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.</p><p><b>CONCLUSION</b>MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).</p>

B-cell lineage study in patients with juvenile idiopathic arthritis

Juvenile idiopathic arthritis [JIA] is the most common rheumatic disease in children. The exact causes of disease are still poorly understood. It seems that B cells have several functions in JIA, including production of autoantibodies, antigen presentation, production of cytokines, and activation of T cells. Here, we aimed to evaluate B-cell lineage and its precursors in the bone marrow of patients with JIA Twenty consecutive patients with JIA were enrolled in this study. JIA is subdivided into three groups of Pauciarticular, Polyarticular, and Systemic JIA. Bone marrow mononuclear cells were separated. Then we analyzed the immunophenotype of the JIA patients by flow cytometry. After separation, the mononuclear cells were stained specific for B cell lineage [CD10, CD19 and CD20], T cell lineage [CD3] and non specific lineage [CD34, HLA-DR and TdT]. Flow cytometric study of bone marrow showed that JIA patients had low level of CD10, CD19, and CD20.Polyarticular patients had lower level of D10, CD19, and CD20 than pauciarticular JIA patients and systemic onset JIA patients had lower levels than both of them. Decreasing of B cell precursor in bone marrow is one of mechanisms for pathogenesis of JIA and the more decreased B cell precursors in bone marrow are, the worst severity of the disease is. Significant differences in CD10 content of bone marrow were detected between the polyarticular and pauciarticular groups. So, it seems that polyarticular JIA patients had lower percentage of pre B cell stage

Blasts in peripheral blood sample of patients with acute leukemia; a comparative study by H1 and flow cytometry

Blast in peripheral blood and bone marrow is a diagnostic factor in acute leukemia, the percentage of blasts will be also important in patient management, particularly after chemotropy. Although conventional study of the cells by light microscopy is an essential step, however it is time consuming and the obtained results may vary from one to other. The H1 system that not only able to automatically differentiate all blood leukocytes, it is also calculate the percentage of blast cells. To evaluate the accuracy of results by the H1 on blast, the study was set to investigate that issus in comparison by CD45 dimly expressed cells [blasts] that computed by flow cytometry. Blood samples from 17 patients with acute lymphoid leukemia [ALL] and 31 patients with acute myeloid leukemia [AML] simultaneously were studied by H1 and flow cytometry using anti-CD4S and CD45/RALS gating. Blood samples from 50 patients with chronic lymphocyte leukemia [CLL] were used as negative controls. The mean values of H1-blasts for B-ALL, T-ALL and AML were 16.3%, 27% and 49.6%, respectively. This figures were 66.6%, 79.7% and 87% by flow cytometry. Both systems were not identified any blast in CLL cases. Analysis of blood samples by H1 in cases with CLL is trusty, but in cases with acute leukemia is unreliable. In comparison to flow cytometry, H1 is identified significantly [p<0.05] lower number of blast in acute leukemia

Determination of laboratory reference ranges for lymphocyte immunophenotype subsets in healthy adult Japanese using flow cytometry.

This study represents a comprehensive evaluation of normative values for lymphocyte immunophenotype subsets using flow cytometry techniques in a Japanese population. Lymphocyte reference ranges were determined for percentage and absolute count of T, B, and NK cells in healthy adult Japanese using an extensive two-color immunophenotyping panel and consistently applied quality control methodology. Reference values were also determined for activation markers on CD3+ lymphocytes CD3+/CD25+, CD3+/CD38+ and CD3+/HLA-DR+. Differences in age and gender were observed for specific lymphocyte subsets. Comparison of the Japanese study with a Thai multi-center study that used similar methodology also demonstrated ethnic differences in lymphocyte reference ranges. The results in this study strongly suggest that reference values derived from studies in one population may not be applied to another population even when similar protocols for reagents, instruments and procedures are used although such studies do appear useful for epidemiological comparisons.

Immunophenotype and ultrastructure of B-cell lymphoproliferative disorder with cytoplasmic projection / 华中科技大学学报（医学）（英德文版）

To identify the knowledge of rare lymphoproliferative disorder, the clinical and biological features of three kinds of lymphoproliferative disorders with cytoplasmic projections were compared. The clinical manifestations, ultrastructure and immunophenotype were analyzed. The results showed that hairy cell leukemia (HCL), splenic lymphoma with villous lymphocyte (SLVL) and hairy cell leukemia-variant (HCL-V) had some common characters including splenomegaly, peripheral blood and bone marrow infiltration by villous lymphocyte and B lymphocyte immunophenotype; but these three disorders had specific features respectively. It was concluded that overall analysis of clinical and laboratory features might be contributive to the differential diagnosis of these three disorders.

Surveying the chronic complications of mustard gas on lymphocyte B by means of surface markers

Mustard gas may cause acute [early] and chronic [late] lesions. Acute lesions include disseminated bullous lesions on skin, respiratory, and GI disorders; chronic complications are chronic bronchitis, severe asthma, and malignancies. B cells are active part of humoral immunity that secrete antibodies to defend our body.We have evaluated 3 groups of patients, each including 25 patients as well as 10 control subjects. Complete blood count, morphological and flowcytometric studies were carried out. For flowcytometric studies 4 markers of CD45, CD19, HLA-DR, and CD25 were assessed on surface of B cells with CD45/CD19/HLA-DR phenotype. Their performance was investigated with CD25. Our findings have revealed a significant decrease in CD25 on B cells' surface that is associated with recurrent microbial infections, in patients exposed to mustard gas. Genetic changes in B cells, due to mustard gas inhalation may predispose individuals to recurrent infections such as pneumonia

Longitudinal follow-up of lymphocyte subsets during the first year of life.

A longitudinal study of lymphocyte subsets during infancy was evaluated by using the flow cytometric immunophenotyping method. Two hundred and thirteen blood samples were obtained from 92 healthy, full-term infants of the following ages: 1-7 days old (n = 43), 3 months old (n = 55), 6 months old (n = 57) and 11 months old (n = 58). The absolute numbers of CD3+ and CD3+/CD4+ T lymphocytes increased from birth to 3 months of age, and remained stable thereafter. The absolute number of CD3+/CD8+ T lymphocytes increased from birth to 11 months of age. The absolute number of CD19+ B lymphocytes and NK cells increased rapidly (3 months) after birth and continued to increase throughout the study period. However, the changes in the relative counts of lymphocyte subsets did not always correspond with the changes in their absolute numbers. These results demonstrate the age-related changes in lymphocyte subpopulations and provide reference ranges for lymphocyte subsets during infancy.

Study of in vitro expansion and differentiation into neuron-like cells of human umbilical cord blood mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the isolation, purification and expansion of human umbilical cord blood mesenchymal stem cells (MSCs) into neuron-like cells in vitro.</p><p><b>METHODS</b>Human cord blood samples were obtained sterilely with 20 U/ml preservative-free heparin. MSCs were isolated by lymphocyte separation medium (density 1.077 g/ml), and purified and expanded with Mesencult trade mark medium. The surface antigen expression of MSCs was detected by flow cytometry. The passage 2, 5 and 8 of the expanded MSCs were induced to differentiate to neuron-like cells. Specific markers and structures were detected by immunohistochemistry and histochemistry methods.</p><p><b>RESULTS</b>The number of MSCs increased two- to three-fold with each expanded passage. 6.6 x 10(5) primary MSCs were expanded ten passages to reach a number of 9.9 x 10(8), and was increased about 1.5 x 10(3)-fold. Flow cytometry showed that MSCs did not express antigens CD(34), CD(11a) and CD(11b), but expressed strongly CD(29) and weakly CD(71), which was identical to human bone marrow-derived MSCs. 70% cells exhibited typical neuron-like phenotype after induction. Immunohistochemistry staining showed that all of the induced different-passage MSCs expressed neurofilament (NF) and neuron-specific enolase (NSE). Special Nissl body was found by histochemistry.</p><p><b>CONCLUSION</b>MSCs in human umbilical cord blood can expand in vitro and differentiate into non-mesenchymal cells.</p>

Study on the immunophenotypes of bone marrow cells from patients with myelodysplastic syndromes and its clinical implications / 中国实验血液学杂志

The phenotypes of the bone marrow cells in various subtypes of myelodysplastic syndromes (MDS) and its clinical implication were explored. The antigen expression of a panel of antigens expressed in marrow cells from 30 patients with subtypes of MDS was assayed by alkaline phosphatase anti-alkaline phosphatase method. The results showed that the expression of myeloid antigens appeared abnormality, CD13 and CD33, found on granulocyte and macrophage precursors, increased, and CD15 decreased. There were no significant changes for monocytic antigen CD14 and lymphoid antigens CD7 and CD10. CD34 was increased in RAEB/RAEB-t and was not increased in RA/RAS patients. CD71, expressed by erythroblast and proliferative cells, was higher in all subtypes of MDS than that in control group. It is suggested that the bone marrow cells from MDS patients showed abnormality of more than two series of immunophenotypes, detection of immunophenotype in MDS cells might be contributed to the diagnosis and predicting prognosis.

Detection of cytoplasmic antigens by flow cytometry and its implication for leukemia immunophenotyping / 中国实验血液学杂志

The purpose of this study was to optimize a fixation procedure for detection of cytoplasmic antigens by flow cytometry(FCM) and to evaluate the effect of intracellular CD3, CD22, CD79a and myeloperoxidase(MPO) in lineage assignment. Four kinds of fixation procedure and three or four color direct immunofluorescence staining were used to permeate cell membrane and label cell surface and intracellular antigens by means of FCM. Results showed that percentage of cytoplasmic antigens positive cells was the highest and cell scatter and fluorescence intensity of CD45 were not changed after using of FACS permeabilization solution. MPO protein was positive in 16/18 acute myeloid leukemia(AML) patients. 4 cases of T cell-acute lymphoblastic leukemia (T-ALL) cases were positive for cytoplasmic CD3(c CD3) but surface CD3 was negative. c CD22 was only detected in 9/13 of B-ALL and cCD79a was positive in 5/5 B-ALL. 18/38 cases of acute leukemia were expressed in more than one lineage marker, 8/21 cases of acute non-lymphocytic leukemia(ANLL) were CD7 positive. 7/17 cases of acute lymphocytic leukemia (ALL) expressed CD13. After further cytoplasmic antigen detection, one was considered to be a T/myeloid biphenotypic leukemia, another one was diagnosed as biclonal or mixed leukemia. The results suggest that intracellular CD3,CD22,CD79a and MPO are lineage specific markers, they are very important for biphenotypic and biclonal/mixed acute leukemia identification

Terminal maturation of resting B cells induced by macrophage factors

Mouse splenic macrophages from BALB/c nude mice (purified by plastic adherence) or cloned macrophage hybridomas stimulated with jacalin (12.5 microg/ml), a D-Gal binding lectin, produce one or more B-cell stimulatory factors which cause splenic B cells from BALB/c or C3H/HeJ mice to secrete immunoglobulin in a polyclonal manner as detected by reverse protein A plaque assays. Jacalin-stimulated macrophage supernatants (JacSup) activate both normal and Percoll gradient-purified small high-density (resting) B cells. Supernatants from total or resting BALB/c spleen cells cultured for 7 days in the presence of JacSup (derived from splenic BALB/c nude mice macrophages) were assayed for immunoglobulin isotypes by ELISA. Resting B cells produce only IgG3 and IgM, whereas total B cells secrete IgG3 and IgM as well as IgG1, IgG2a, IgG2b and IgA. Resting and total B cells from BALB/c nude mice are also stimulated by macrophage supernatants to secrete immunoglobulin, thus indicating that this activity is likely to be T cell independent. Moreover, jacalin-stimulated macrophage supernatants did not induce spleen cells or purified B cells to proliferate. Fractionation of factor-rich supernatants on a Sephacryl S-200 column revealed that the factor activity is located in fractions corresponding to a molecular mass of 25-27 kDa. Taken together, these results suggest that upon the action of a macrophage factor(s) resting B cells undergo terminal differentiation without proliferation in the absence of T cells.

A clinicopathologic study on the diffuse malignant lymphoma: a morphologic and immunophenotypic analysis in 62 patients at Harbor-UCLA Medical Center

In order to compare the prognoses of patients with diffuse malignant lymphomas on the basis of histology and immunophenotypes, we retrospectively studied 62 cases of diffuse lymphoma arising in lymph nodes. We also evaluated the reactivity patterns of monoclonal antibodies (MoAb) LN1, LN2 and LN3 to determine the criteria for making a differential diagnosis in B cell lymphomas. The immunologic phenotypes were determined by the avidin biotin peroxidase complex method, using frozen or paraffin fixed tissues. The majority (66.3%) were B cell with the remaining 20.9% being T cell and 12.9% were non-B, non-T cell lineage. Immunological heterogeneity was found especially in the mixed small and large cell and the immunoblastic lymphomas. There was no significant difference between B- and T-cell lymphomas with respect to survival and death (P > 0.05). Histologically 79% (49/62) of the lymphoma was large cell and 21% (13/62), small cell lymphoma. There was a difference in prognosis between low, intermediate and high-grade of lymphomas. However there were no significant differences among the subtypes of the diffuse aggressive lymphomas. Factors associated with poor prognosis were advanced stages (P < 0.025) and histology of the malignant lymphomas. MoAb LN1, LN2 and LN3 gave positive staining in 83.3%, 91.7% and 60% of B cell lymphomas, respectively. The most common phenotypic pattern in B cell lymphomas was LN1+, LN2+, LN3+/-, suggestive of follicular center cell origin. As a panel, phenotypic patterns of MoAb LN1, LN2 and LN3 may be useful in differentiation of follicular center cell lymphoma from others.

Differentiation antigen on murine B lymphocytes defined by monoclonal antibodies.

Spleen cells from an AKR/J X DBA/2J F1 mouse immunized with NZB/BIN spleen cells were fused with SP2/0-Ag14. Two hybrid cell lines, B220-1 and B220-2, were established that secreted antibody to the B-lineage specific B220 antigen. B220-1 and B220-2 are present on 45-55% of splenic and bone marrow lymphocytes and absent from thymus. By flow cytometry, all immunoglobulin-bearing cells were stained by these monoclonal antibodies. Although these monoclonals do not stain thymocytes, they do react weakly with Lyt-2+ peripheral T cells. Dual parameter analysis of B lymphocytes using RA3-3A1 or 14.8 show that these monoclonals recognized the same population. Prior incubation with RA3-3A1 or 14.8 was unable to completely block the binding of B220-1 or B220-2, implying that the epitopes recognized are different from the previously described monoclonal antibodies. Immunoprecipitation of the splenic lymphocyte reveals a molecule which migrates on SDS-PAGE as a single band with MW of 220,000 daltons. Expression of the distinct antigens recognized by B220-1 and B220-2 varied among mouse strains, indicating previously unappreciated polymorphism of the B220 molecule. These monoclonals are useful for cytotoxic elimination of B cells and for three-color flow cytometry.