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In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20 + CD27 + IgD - isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers., (©2021 The Authors; Published by the American Association for Cancer Research.)

Ofatumumab (OFA), a human CD20-targeting mAb, kills B lymphocytes using the innate immune system including complement-dependent cytotoxicity (CDC). The efficacy of OFA in patients with chronic lymphocytic leukemia (CLL) is limited by drug resistance, which is not well characterized. To better understand mechanisms of resistance, we prospectively studied CLL cells isolated from blood samples collected before and after in vivo exposure to the initial dose of OFA therapy in 25 patients undergoing their first treatment for progressive CLL. As previously reported, OFA therapy rapidly decreased the absolute lymphocyte count, CD20 expression by CLL cells, and serum complement levels. We now show that after administration of the first dose of OFA, there was a modest rebound in the absolute lymphocyte count and serum complement levels, but substantial ongoing loss of CD20 expression by CLL cells. These post-OFA treatment CLL cells were highly resistant to OFA-mediated CDC but retained sensitivity to alemtuzumab-mediated CDC in vitro. Posttherapy serum OFA levels correlated inversely with both the amount of pretreatment circulating cell-bound CD20 and with the decrease in this value following treatment. In vitro OFA-mediated CDC did not predict clinical responses, and the patients with first-dose reactions to OFA did not have markers of increased complement activation in vivo. We propose that optimal efficacy of CD20- targeted therapy for CLL requires determining an mAb dose size and frequency that optimizes CLL killing without exceeding the capacity of the cytotoxic mechanisms and thus minimizes loss of CD20 expression in the surviving CLL cells.

Angioimmunoblastic T-cell lymphoma (AITL) exhibits a multifaceted clinical picture and distinct architectural patterns that correlate with disease progression and the number of neoplastic cells. In this study we investigated the expression of the transcription factor T-bet and correlated it with the architectural patterns in 29 cases of AITL. Double immunolabelings for T-bet, CD20, CD3 or PD1 revealed the following patterns: predominant T-bet expression by neoplastic T-cells (A), by aggregates of small B-cells (B) or by B-immunoblasts (C). The majority of cases of AITL pattern II showed a T-bet expression pattern B (6/8 cases), while the majority of those with pattern III exhibited the T-bet pattern A (11/21 cases). We propose that T-bet expression by B-cells represents a T-cell independent immune response trying to cope with opportunistic infections, while T-bet expression by neoplastic T-cells is linked to the introduction of a Th17 response responsible for the immunologic derangements characteristic of AITL.

Background: Tumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC)., Methods/results: TiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23(+), CD80(+)) and produced immunoglobulin (Ig; IgG secretion in 55% of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigen-experienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV., Conclusion: In summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specific antigens.

To dissect the mechanisms of anti-TNFα-induced autoimmunity we examined the phenotype and function of B cells from anti-TNFα-treated patients. Levels of Lyn, Syk, SHP-1, tyrosine 348 phospho-Syk (Y348-Syk) and tyrosine phosphorylated (P-Y) proteins were evaluated and B-cell-surface CD20, CD21 and CD5 were also assessed in 29 patients treated with TNF-α blockers. Following treatment, Lyn, but not Syk or SHP-1, significantly increased particularly in patients with spondyloarthropathies. Increased Lyn levels following treatment correlated with increased Lyn activity as evidenced by a 2.9-fold increase of Y348-Syk (a Lyn target). Peripheral B-cells from 56.3% of the patients displayed a tendency towards increased P-Y levels without any BCR-initiated activation during treatment. CD20, but not CD21, significantly increased in patients with rheumatoid arthritis. Circulating CD5+ B-cells were also significantly expanded during treatment. Our findings suggest that B cells in anti-TNFα-treated patients display functional and phenotypical aberrations that may enhance our understanding of TNF-α blocker-induced autoimmunity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Primary cardiac lymphoma (PCL) is a rare and fatal disorder. It may often mimic other common cardiac tumors like cardiac myxoma because of similarities in the clinical presentation. We report a case of PCL of diffuse large B-cell type, in a 38-year-old, immunocompetent male who presented with superior vena cava syndrome that was excised as a myxoma. Histology revealed a large cell population diffusely and strongly expressing CD45, CD20, MUM1/IRF4 and FOXP1 hinting at an activated B-cell (ABC)-like phenotype. After four cycles of Rituximab with CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisolone) the tumor regressed completely but the patient had a relapse and subsequently succumbed to the disease confirming the aggressive nature. The aggressive behavior of PCL may be possibly linked to its ABC-like origin.

Chronic lymphocytic leukemia is less effectively treated than other B cell malignancies with the anti-CD20 agent, rituximab, presumably due, at least in part, to low CD20 expression. CD20 expression is typically measured by flow cytometry, which may not be quantitative. This study was undertaken to measure total CD20 protein in CLL B cells using quantitative immunoblot analysis. The results demonstrated that total CD20 protein levels were consistently decreased by ∼60% in CLL B cells with low CD20 fluorescence staining. Surprisingly, real-time polymerase chain reaction analysis showed that CD20 mRNA levels were normal or close to normal, depending on the comparative B cell population, and did not correlate well with protein expression. We conclude that CD20 protein is substantially decreased in CLL due to a post-transcriptional defect., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: There is an increasing evidence that the presence of CD20 B cells is associated with poor clinical outcomes in acute cellular rejection (ACR), but clinical significance of CD38 B cells is undetermined. We attempted to examine the clinical significance of the CD38 B cells alone or in combination with CD20 B cells in renal transplant recipients with ACR., Methods: Fifty-four patients with ACR were included. Biopsy specimens were stained for CD20 and CD38. The clinical outcomes of CD20 or CD38 B cells were evaluated with late-onset and repeated ACR, steroid resistance, incomplete recovery after rejection treatment, and allograft survival., Results: Twenty-three patients (42.6%) had CD20 and 25 (46.3%) patients had CD38 B cells. Of these, 15 patients (27.8%) were positive for both CD20 and CD38 (CD20CD38). CD38 patients had higher rates of late-onset or repeated ACR and incomplete recovery compared with CD38 patients (P<0.05). The patients with CD20CD38 had a higher incomplete recovery rate than did patients with only CD20 or CD38 (P<0.05). The 5-year allograft survival was lower in CD20 and CD38 patients than in CD20 or CD38 patients (P<0.05 for each). CD20CD38 patients had lower graft survival than did patients with CD20 or CD38 alone (P<0.05)., Conclusion: Infiltration of CD38 B cells alone or in combination with CD20 B cells is a predictor for poor clinical outcomes of ACR in renal allograft.

B lymphocyte infiltration in renal acute allograft rejection has been associated with steroid resistance and poor outcomes. We aimed to measure CD20 mRNA in urine of renal transplant patients with graft dysfunction and correlate with the histological diagnosis and immunohistochemical (IH) staining for CD20. A total of 48 urine samples were analyzed (21 with acute rejection, 10 with chronic allograft nephropathy, 11 with unspecific tubular lesions, 3 with acute pyelonephritis and 3 with polyomavirus nephropathy). Higher urinary CD20 levels associated with a positive IH staining for CD20 (>50 positive cells/HPF) in renal tissue (p=0.04), with a sensitivity of 83.3% and a specificity of 51.6%. Within the acute rejection group, a positive staining for CD20 was not associated with graft loss, steroid resistance or lack of return to basal creatinine after treatment, but was associated with higher serum creatinine at 3 and 6 months, 1 and 2 years after the acute episode (p<0.05). In conclusion, we showed that urinary levels of CD20 detected by RT-PCR had a high sensitivity for CD20+ staining in the corresponding renal tissue, but with a low specificity. Patients with clusters of CD20+ cells >50/HPF had higher serum creatinine after 2 years of follow up.

To assess the significance of B-cell and plasma cell infiltrates in renal allografts, we compared expression of B-cell-associated transcripts (BATs) and immunoglobulin transcripts (IGTs) to histopathology and function in 177 renal allograft biopsies for clinical indications. BAT and IGT expression correlated with immunostaining for B cells and plasma cells and with expression of B-cell and plasma cell transcription factors. BATs and IGTs were increased in both T-cell-mediated and antibody-mediated rejection. BAT and IGT scores were strongly related to time posttransplant: biopsies <5 months expressed less BATs and did not express increased IGTs. In contrast, T-cell-associated transcripts were independent of time posttransplant. In biopsies > or =5 months, BAT and IGT scores correlated with interstitial inflammation, tubular atrophy and interstitial fibrosis. By regression tree analysis, the only variables independently correlated with BATs and IGTs were time and inflammation. Expression of BATs and IGTs correlated with renal function, but this relationship was due to differences in early versus late biopsies: BATs and IGTs were not related to function or future function after correcting for time.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. Although latent HHV-8 DNA can be detected in B cells from persons with these cancers, there is little information on the replication of HHV-8 in B cells. Indeed, B cells are relatively resistant to HHV-8 infection in vitro. We have recently shown that DC-SIGN, a C-type lectin first identified on dendritic cells (DC), is an entry receptor for HHV-8 on DC and macrophages. We have also demonstrated previously that B lymphocytes from peripheral blood and tonsils express DC-SIGN and that this expression increases after B-cell activation. Here we show that activated blood and tonsillar B cells can be productively infected with HHV-8, as measured by an increase in viral DNA, the expression of viral lytic and latency proteins, and the production of infectious virus. The infection of B cells with HHV-8 was blocked by the pretreatment of the cells with antibody specific for DC-SIGN or with mannan but not antibody specific for xCT, a cystine/glutamate exchange transporter that has been implicated in HHV-8 fusion to cells. The infection of B cells with HHV-8 resulted in increased expression of DC-SIGN and a decrease in the expression of CD20 and major histocompatibility complex class I. HHV-8 could also infect and replicate in B-cell lines transduced to express full-length DC-SIGN but not in B-cell lines transduced to express DC-SIGN lacking the transmembrane domain, demonstrating that the entry of HHV-8 into B cells is related to DC-SIGN-mediated endocytosis. The role of endocytosis in viral entry into activated B cells was confirmed by blocking HHV-8 infection with endocytic pathway inhibitors. Thus, the expression of DC-SIGN is essential for productive HHV-8 infection of and replication in B cells.

CD20 antibody depletion of B lymphocytes effectively ameliorates multiple T cell-mediated autoimmune diseases through mechanisms that remain unclear. To address this, a mouse CD20 antibody that depletes >95% of mature B cells in mice with otherwise intact immune systems was used to assess the role of B cells in CD4(+) and CD8(+) T cell activation and expansion in vivo. B cell depletion had no direct effect on T cell subsets or the activation status of CD4(+) and CD8(+) T cells in naive mice. However, B cell depletion impaired CD4(+) T cell activation and clonal expansion in response to protein antigens and pathogen challenge, whereas CD8(+) T cell activation was not affected. In vivo dendritic cell ablation, along with CD20 immunotherapy, revealed that optimal antigen-specific CD4(+) T cell priming required both B cells and dendritic cells. Most importantly, B cell depletion inhibited antigen-specific CD4(+) T cell expansion in both collagen-induced arthritis and autoimmune diabetes mouse models. These results provide direct evidence that B cells contribute to T cell activation and expansion in vivo and offer insights into the mechanism of action for B cell depletion therapy in the treatment of autoimmunity.

Background: Intrarenal B cell clusters are associated with poor clinical outcome in acute interstitial rejection. The incidence of B cell aggregates in vascular rejection and the effect of therapy with the monoclonal CD20 antibody rituximab on intrarenal B cells are currently unclear., Methods: We analyzed the incidence of B cell clusters in patients with vascular rejection by immunohistochemistry and compared the influence of rituximab treatment plus conventional therapy with that of conventional immunosuppression alone on intrarenal B cells. Furthermore intrarenal expression of the B cell attracting chemokine BCA-1/CXCL13 and the lymphoid chemokine SLC/CCL21 were analyzed., Results: Nine of 16 patients with vascular rejection displayed intrarenal B cell clusters strictly co-localizing with expression of the B cell attractant chemokine BCA-1/CXCL13. Addition of rituximab to conventional treatment lead to complete depletion of intrarenal B cells (98.3+/-136.4 CD20, 90.7+/-113.2 CD19 vs. 0+/-0 CD20, 0+/-0 CD19 B cells/hpf, P<0.001). Creatinine decreased from 5.0+/-4.1 to 1.9+/-0.4 mg/dl at discharge, and to 1.9+/-0.5 mg/dl after three months (P<0.02). No effect on intrarenal B cells was observed in the patients not treated with rituximab (72.8+/-73.0 vs. 80.3+/-75.3 CD20, 75.6+/-86.6 vs. 85.7+/-82.0 CD19). At discharge, creatinine had improved in this group from 5.1+/-4.1 mg/dl to 1.8+/-0.5 mg/dl and to 1.7+/-0.6 mg/dl after 3 months (P<0.05)., Conclusion: In summary, our study reports two main findings, namely the previously unrecognized high prevalence of intrarenal B cell clusters in 56% of biopsies with acute vascular rejection and a complete depletion of intrarenal B cells by addition of Rituximab to conventional treatment.

B cells in patients with rheumatoid arthritis (RA) are hyperactivated. Although B cell receptor signal transduction may be affected by various response regulators, CD22 plays an important role as a response regulator of B cells. Therefore, we investigated and examined CD22 expression on peripheral blood B cells of patients with RA. Thirty-two RA patients and 16 controls were enrolled in this study, and CD22 expressions on B cells were analyzed by flow cytometry. In patients with RA, CD22(+) B cells significantly decreased in comparison to the controls (ratio: P < 0.05). However, there was no correlation between this decrease and the clinical data. Interestingly, CD5(+) CD22(-) B cells significantly increased in RA patients. The decrease in CD22(+) B cells and increased in CD5(+)CD22(-) B cells play critical roles in the pathogenesis of RA mediated by the activation of B cells.

Although differentiated cells normally retain cell-type-specific gene expression patterns throughout their lifetime, cell identity can sometimes be modified or reversed in vivo by transdifferentiation, or experimentally through cell fusion or by nuclear transfer. To examine the epigenetic changes that are required for the dominant conversion of lymphocytes to muscle, we generated heterokaryons between human B lymphocytes and mouse C2C12 myotubes. We show that within 2 days of heterokaryon formation lymphocyte nuclei adopt an architecture resembling that of muscle and then initiate the expression of muscle-specific genes in the same temporal order as developing muscle. The establishment of this muscle-specific program is coordinated with the shutdown of several lymphocyte-associated genes. Interestingly, erasing lymphocyte identity in reprogrammed cells requires histone deacetylase (HDAC) activity. Inhibition of HDAC activity during reprogramming selectively blocks the silencing of lymphocyte-specific genes but does not prevent the establishment of muscle-specific gene expression. Successful reprogramming is therefore shown to be a multi-step process in which the acquisition and extinction of lineage-specific gene programs are separable events.

Membranous glomerulonephritis (MGN), histologically defined by subepithelial immune deposits, is the most common cause of nephrotic syndrome in Caucasian adults. The current hypothesis of the underlying disease mechanism postulates production of antibodies against podocyte-derived antigens. Respective antigens could be demonstrated in different animal models and recently in human neonatal MGN. Further support for this hypothesis was generated by the response of human MGN to therapeutic B cell depletion by rituximab. However, the role of B cells in this disease is not well defined. In this study, the interstitial expression of CD20 mRNA was determined in 31 MGN patients and controls (tumor nephrectomies (n = 4), minimal change disease (MCD, n = 10) and focal segmental glomerulosclerosis (n = 6)). CD20 mRNA expression was significantly higher in MGN patients compared to controls. By immunohistochemistry, a focal or diffuse interstitial B cell infiltration could be detected in MGN patients (n = 63), which was absent or minimal in patients with MCD (n = 11). These data suggest an involvement of B cells in the pathogenesis of MGN, possibly as antigen-presenting cells. Further studies should investigate the potential to predict the response to therapeutic B cell depletion by intrarenal CD20 quantification, a potential diagnostic basis for the selection of a specific therapy currently evolving for renal disease.

B-cell chronic lymphocytic leukemia (CLL) has been traditionally described as a disease characterized by an accumulation of quiescent small lymphocytes with decreased susceptibility to apoptotic cell death. However, small numbers of "atypical" lymphocytes and prolymphocytes (PL) are frequently observed in the bone marrow (BM) of patients with CLL. In this study, we examined BM biopsy and aspirate specimens obtained from seven patients with atypical CLL. Using a double labeling (Ki-67+/CD20+) immunohistochemical method, we found that an appreciable number of the atypical CLL cells expressed the proliferation-associated protein Ki-67. Because CLL is characterized by a slow change in the peripheral blood (PB) lymphocyte count, we reasoned that a subpopulation of CLL cells probably undergoes spontaneous apoptosis. Using Western blot analysis, we observed expression of procaspase-9, procaspase-10, and poly(ADP-ribose) polymerase by the neoplastic cells in all seven cases of CLL, and procaspase-3 and procaspase-8 expression in six neoplasms. We also detected cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in four and five CLL cases, respectively. To determine whether CLL cells undergo spontaneous apoptosis, we performed the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using BM biopsy specimens. We found TUNEL-positive lymphocytes in areas infiltrated by CLL. In summary, our data show that subpopulations of B-lymphocytes are proliferating or undergoing spontaneous apoptotic cell death in patients with atypical CLL.

Purpose: To investigate potential immunotherapeutic strategies in B lymphocytic malignancies we looked for CTLs recognizing CD19 and CD20 epitopes., Experimental Design: Three CD19 and CD20 peptides binding to HLA-A*0201 were identified and used to detect peptide specific CTLs by a quantitative real-time PCR to measure IFN-gamma mRNA expression in 23 healthy individuals and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 follicular lymphoma, 2 acute lymphocytic leukemia, and 1 large cell lymphoma). Peptide-specific CTLs were expanded in culture with CD40-activated B cells to test lytic activity in three patients., Results: In healthy individuals, CD8+ T-cell responses were detected in one to CD19(74-82), in three to CD20(127-135), and three to CD20(188-196). Seven of 27 patients (6 with CLL) had CD8+ T cells recognizing CD19(74-82). Seven patients responded to CD20(127-135) and three to CD20(188-196). All were CLL patients. CD19(74-82)-specific CTLs from three patients were expanded over 4 weeks. These cells were HLA-A*0201 specific and lytic for peptide-loaded antigen-presenting cells but not to malignant or unpulsed B cells., Conclusions: CTLs that recognize CD19 and CD20 epitopes exist in healthy individuals and may be increased in CLL patients. They are of low avidity and require high doses of peptide for activation. Strategies to increase T-cell avidity would be necessary for T-cell immunotherapeutic approaches using the peptides studied.

Pyothorax-associated lymphoma (PAL) develops decades after receiving artificial pneumothorax for pulmonary tuberculosis. The lymphomas, develop in tissue affected by long-standing severe inflammatory process. Most cases demonstrate diffuse large B-cell lymphoma. We present a patient with T-cell phenotype-positive and B-cell phenotype-negative (CD7+, CD43+, CD19-, and CD20-) PAL. Southern blot hybridization using immunglobulin heavy chain J region (IgH) gene probe revealed a monoclonal rearrangement, and hybridization using T-cell receptor beta chain (TCR) gene probe revealed a germline configuration. This indicates that the tumor origin was of B-lymphocytes. Chromosomal abnormality of the lymphoma was complicated. It suggested that many transformations occurred. In the transformation process, probably B-cell antigens were lost, and T-cell antigens were aberrantly expressed.

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8(+) cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th(1)/Th(2) balance to Th(2) dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th(2) cells., (Copyright 2002 Elsevier Science Ltd.)

Helicobacter pylori infection has been implicated in the development of gastrointestinal malignancy in adults and children. The histopathological processes that lead to such development are unknown. We compared the immune cell repertoire of mucosal lymph follicles in children with H. pylori infection to B cell type mucosal associated lymphoid tissue (MALT)-lymphoma of adults. The B and T cell populations residing within the lymph follicles and/or within B cell type MALT lymphoma were characteriZed by an immunohistochemical technique, utilizing B and T cell markers including: CD3, CD4, CD8 (T cells); CD20, CD40, GD74, BLA36, CD80, CD86 (B cells). Stain intensity was compared between the samples. T cell repertoire was observed within the lymph follicles, but not in the B cell MALT-lymphoma specimens. No significant difference was observed between the staining of CD40, CD74, CD8, and BLA36. The B cell markers, CD80 and CD86, were found within the centrocytic zone of the lymph follicle. In the B cell repertoire, no significant difference was observed between the lymph follicles of children with H. pylori infection and the adult MALT-lymphoma specimens except in CD20. B and T cells were in close anatomical proximity, enabling them to interact and exchange immunological information.

The CD20 surface antigen is expressed on the great majority of B-cell lymphoma cases. The expression of this antigen ranges from moderate to bright, and it is neither internalized nor shed. These characteristics make CD20 a common target for antibody directed lymphoma therapy. The development of strategies to significantly increase CD20 expression on lymphoma cells is therefore of great interest as a means of increasing specific targeting and cell kill in antibody therapy and radio-immunotherapy. We present here data demonstrating that relatively low doses of external beam radiotherapy are capable of significant and consistent increases in CD20 surface expression in vitro. The effect is dose related up to approximately 10 Gy and is maximal in the first day after radiotherapy. We believe that these data may suggest a potent way to combine a short pretreatment course of external beam radiotherapy with a subsequent course of immunotherapy using either an unlabeled antibody or a radio-immunotoxin., (Copyright 2001 Wiley-Liss, Inc.)

Most antibody panels proposed for flow cytometric immunophenotyping of non-Hodgkin's lymphomas and chronic lymphoid leukemias include anti-CD20 and FMC7 antibodies. As in our experience, reactivity of B-cells with these antibodies seemed to be correlated, we evaluated whether the simultaneous use of anti-CD20 and FMC7 antibodies is justified. Using flow cytometry, we measured the binding of these 2 antibodies to the B-cells of 67 bone marrow aspirates, 31 lymph node biopsies, 18 peripheral blood specimens, and 12 tissue samples from other locations. The diagnoses included 50 cases without overt abnormalities, 5 reactive lymphadenopathies, 56 lymphomas and chronic lymphoid neoplasias, and 17 cases with other malignancies. Although CD20 expression was consistently higher, we observed a significant and strong correlation between CD20 and FMC7 antigen expression on B-lymphocytes, irrespective of the nature of the sample or disease (r=0.910; P < 0.001). Moreover, FMC7 antigen expression on B-cells could be predicted by CD20 expression with a sensitivity of 96%, a specificity of 94% and an efficiency of 96%. Our results show that although differing in intensity, expression of CD20 on B-cells closely parallels that of FMC7 antigen. We, therefore, conclude that little additional information is revealed by using FMC7 in immunophenotyping of non-Hodgkin's lymphomas or chronic lymphoid leukemias if intensity of CD20 expression is taken into consideration.

The novel human pre-B cell line OZ was established from a patient with an aggressive form of non-Hodgkin's lymphoma. Karyotypic analysis of both the primary tumour and OZ cells revealed several marker chromosomes, including the t(14;18)(q32;q21) translocation, which involves the Bcl-2 gene, and alterations on chromosome 17p. Southern blot analysis found identical rearrangements in the 5' region of Bcl-2 gene in the primary tumour and OZ cells. Homozygous deletions of the p15INK4B and p16INK4A genes, however, were present only in OZ cells. Western blot analysis detected aberrant small molecular-weight p53 proteins in both cell types. In addition, OZ cells no longer expressed the CD20 antigen. These findings suggest that Bcl-2 gene rearrangement and aberrant p53 expression resulted in the original B-cell tumour. A subsequent transforming event involving the p15INK4B and p16INK4A genes may have generated more immature cells with a growth advantage during in vitro culture. The genetic alterations involving p53, p15INK4B, and p16INK4A may be implicated in the aggressive form of t(14;18)(q32;q21)-bearing tumours and their poor prognosis.

We have previously shown that dual occupancy of sigM and CD40 - essential receptors in T-dependent B cell responses - by antibodies held on CD32-L cells results in the rapid proliferation of resting human B lymphocytes in a cytokine-independent manner. Here we report the detailed phenotype of the blast population emerging in such cultures. By 3 days the levels of CD19 and CD20 have increased 4- and 2-fold respectively: such high level expression of these two pan-B markers is characteristic of cells of germinal centre (GC) origin. B cells co-stimulated via sIgM and CD40 express low level CD23 and almost half become CD5(+); they also acquire CD38 and - importantly - CD77, both of these being selective markers of GC B cells. Expression of sigM and IgD is down-regulated on these cells and a minor, but significant, population of IgG+ cells appears. In marked contrast to GC B cells, the population proliferating in response to dual occupancy of sigM and CD40 has up-regulated and strongly expresses CD44. Morphologically, the cells are heterogeneous but there is a dominant blastic cell type with relatively scanty cytoplasm and having multiple nucleoli, both of which are characteristic of centroblasts; nevertheless, these cells remain morphologically distinct from freshly isolated GC B cells and do not show hallmark features of centrocytes. Although there is substantial cell death occurring by days 6-7 in these cultures, there is no morphological evidence for apoptosis. Thus, the proliferating population that emerges from the dual engagement of antigen receptor and CD40 on resting B cells appears to be bestowed with some features of GC B cells but has others which are incompatible with that particular stage of differentiation. The possibility that it might represent (i) a blast stage that is transitional between activation in T zones and entry into the follicle or (ii) a precursor population that colonizes the primary follicle prior to GC formation is discussed.

Human interleukin-12 (IL-12) is a 70-kDa polypeptide that activates human natural killer cells. It has been purified from the culture supernatant of a human Epstein-Barr virus-transformed B cell line and cloned. We show that native as well as recombinant IL-12 promoted growth of Staphylococcus aureus Cowan I strain (SAC) or anti-mu antibody-activated B cells in a dose-dependent manner. IL-12 also acted in synergy with IL-2 in growth and differentiation of SAC-activated B cells. Since anti-interferon (IFN)-gamma antibody completely abrogates B-cell growth factor (BCGF) activity of IL-12, the BCGF activity is mediated by IFN-gamma. This conclusion is clearly supported by the results that IL-12 indeed induced IFN-gamma production by activated B cells. These results suggest that the B cell proliferative effect of IL-12 may be mediated by autocrine IFN-gamma.