CD40 expression in Hodgkin's disease.

CD40 is a transmembrane protein that belongs to a superfamily of proteins related to nerve growth factor receptor. CD40 is expressed on B cells and some B cell malignancies. It appears to be involved in B cell proliferation and the prevention of apoptosis in germinal center cells, which is accompanied by expression of bcl-2. Its expression is up-regulated by the EBV protein latent membrane protein-1 and cytokines interleukin-4 and interferon-gamma. The expression of CD40 in 37 cases of Hodgkin's disease and 23 cases of non-Hodgkin's lymphoma (11 T cell lymphomas and 12 B cell lymphomas) was examined by paraffin section immunohistochemistry using the BB-20 monoclonal antibody. In 26 of 37 cases of Hodgkin's disease the Reed-Sternberg cells showed strong membrane or cytoplasmic expression of CD40. Only 3 of 23 non-Hodgkin's lymphomas showed any expression of CD40 and then only weakly. There was no correlation between expression of bcl-2 or latent membrane protein-1 with CD40 expression. These results show that there is probable hyperexpression of CD40 in Hodgkin's disease and suggest that dysregulation of CD40 expression may play a role in the pathogenesis of Hodgkin's disease.     

Expression of B7 (CD80) and CD40 antigens and the CD40 ligand in Hodgkin''s disease is independent of latent Epstein—Barr virus infection

Aim—To examine the expression of CD40 and B7 (CD80) antigens and the CD40 ligand in Hodgkin's disease.     

High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of HIV-associated Hodgkin's disease.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease, with an frequency of 15 to 50% in the immunocompetent host. We studied 12 formalin-fixed, paraffin-embedded cases of Hodgkin's disease occurring in human immunodeficiency virus-infected individuals to determine the frequency of EBV in Hodgkin's disease from this population. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated anti-sense oligonucleotide complementary to the EBER1 gene of EBV. EBV RNA was found in the Reed-Sternberg cells and variants in 11 of 12 cases. Double-labeling studies confirmed the presence of EBV RNA in CD15-expressing Hodgkin's cells in all 11 cases, although rare B lymphocytes coexpressing EBV RNA and CD20 were also noted in these cases. The Hodgkin's cells in all 11 EBER-positive cases expressed latent membrane protein. The one case negative for EBV RNA showed the histology of nodular, lymphocyte predominance, a subtype thought to be distinct from other types of Hodgkin's disease.     

The role of CD30, CD40 and CD95 in the regulation of proliferation and apoptosis in classical Hodgkin's lymphoma

AIMS: CD30, CD40 and CD95 are members of the tumour necrosis factor receptor superfamily. Ligation to their respective ligands (CD30L, CD40L, CD95L) will generate a diverse set of signalling cascades. We aim to study the expression pattern of CD30, CD40 and CD95 in classical Hodgkin's lymphoma (cHL) and to correlate the expressions with proliferation and apoptosis in the Hodgkin/Reed-Sternberg (H/RS) cells of cHL with or without associated Epstein-Barr virus (EBV) infection. METHODS: A total of 66 cHL cases were retrieved from the archives. Expressions of CD30, CD40, CD95 and proliferation by Ki-67 expression were detected with an immunohistochemical staining method. Apoptosis index was assessed by in situ TUNEL staining technique on 30 randomly selected cases and the presence of EBV was determined by EBER in situ hybridisation. REUSLTS: Expression of CD30, CD40 and CD95 in the H/RS cells was observed in a high proportion of the cases (100, 93.9, 90.5%, respectively). There was no significant association or correlation of the expression of these molecules with the presence of EBV. Expression of CD40 was associated with expression of the proliferation marker Ki-67 (P=0.044), whereas strong (intermediate and high) expression of CD30 showed a significant correlation with proliferation in the EBV-negative cases only (P=0.025). No correlation was observed for the expression of CD30 and CD40 with apoptosis of the H/RS cells. The childhood cases showed weaker CD95 expression in the H/RS cells than the adult cases, and the expression of CD95 was weaker than that of CD40 in the childhood group. CONCLUSIONS: Our results showed that CD30, CD40 and CD95 are highly expressed in the H/RS cells of the majority of cases of cHL. The expression patterns seem to be independent of EBV and do not correlate with apoptosis of the H/RS cells.     

Varied B-cell immunophenotypes of Hodgkin/Reed-Sternberg cells in classic Hodgkin's disease

AIMS: Recent analyses have suggested that Hodgkin's or Reed-Sternberg (HRS) cells in most cases of classic Hodgkin's disease are derived from germinal centre B-cells. However, there is controversy over which B-cell antigens are expressed in HRS cells. METHODS AND RESULTS: We studied 51 cases of classic Hodgkin's disease to immunohistochemically characterize HRS cells for pan-B-cell markers and specific markers for plasma cells. HRS cells expressed CD20 (L26) in 18 cases (35%), CD19 (B4) in nine (18%) and CD79a (mb-1) in 13 (25%). Furthermore, HRS cells were positive for CD138 (B-B4) in 24 cases (45%) and for PCA-1 in 24 (45%). In 41 (80%) cases, HRS cells expressed more than one B-cell marker. We then subclassified cases into those positive for plasma cell markers (n = 27) (group 1) and those negative for them (n = 24) (group 2). The average age in group 1 (40 years) was younger than in group 2 (54 years) (P < 0.05). The percentage of nodular sclerosis (NS) subtype in group 1 (52%) was 1.5 times greater than in group 2 (33%) (P < 0.05). With regard to Epstein-Barr virus encoded small RNA (EBER) in-situ hybridization, 14 cases (64%) were positive in group 2, but only seven cases (31%) were positive in group 1 (P < 0.025). CONCLUSION: In most cases of classic Hodgkin's disease, HRS cells expressed later stage of B-cell development. We consider that two different clinicopathological groups may correlate with the two different expressions of B-cell markers.     

CD40–CD40Ligand interactions and their role in cytotoxic T lymphocyte priming and anti-tumor immunity

Tumor–specific immunity relies on interactions with the antigen receptors as well as costimulatory molecules, such as those of the CD28/B7 pathway and relatives of the TNFR gene family. Cytotoxic T lymphocytes specific for cellular antigens are in general primed by professional antigen–presenting cells that indirectly present antigens derived from cells in the periphery. This cross–priming of CD8⁺T cells requires signals provided by CD4⁺T helper cells. Although this dependency on [help’ for efficient cytotoxic T lymphocyte priming has been well documented, it was only recently that more mechanistic insight into the nature of this event has been obtained. In the absence of the CD4⁺T cells, signalling through CD40 can replace `help' required for priming of these CD8⁺T cells. These observations indicate that T cell help for cytotoxic T lymphocytes is mediated by CD40–CD40Ligand (L) interactions, most likely through activation of professional antigen–presenting cells that cross–present cellular antigens to these T cells.     

Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals.

Normal nasopharyngeal tissues from 23 individuals who died of causes unrelated to the upper respiratory system and had no evidence of Epstein-Barr virus (EBV)-related diseases were studied using in situ hybridisation (ISH) and immunohistochemistry for the detection of EBV RNA and expression of EBV proteins, respectively. ISH using ³⁵S-labelled riboprobe for EBV EBER RNA showed occasional to a few EBER + lymphocytes in the stroma of nasopharyngeal mucosa in 14/16 cases with available paraffin-embedded tissues. In addition, very rare intraep-ithelial EBER+ lymphocytes were also detected in 3/16 cases. However, in none of these cases was EBER detected in the epithelial cells. Similar results were obtained using a nonradioactive ISH method for EBER (Dako). In 3/23 cases, im-munostaining using monoclonal antibodies for EBV proteins on cryostat sections showed occasional cells in the stroma expressing EBV nuclear antigen 2 (EBNA2), latent membrane protein-1 (LMP), and switch protein encoded by BZLF1 gene (ZEBRA) in two cases and only very rare LMP+ and ZEBRA+ cells in one other case. Double immuno-staining combining alkaline phosphatase anti-alkaline phosphatase (APAAP) for CD markers and indirect immunofluorescence for LMP showed that the LMP+ cells were either CD19+ or less frequently CD3+, but none were CD68+. These results show that both B and T lymphocytes harbouring EBV can be found in the normal nasopharyngeal tissues. Interestingly, EBV proteins associated with lytic viral replication—diffuse early antigen (EA-D), viral capsid antigen (VCA), or membrane antigen (MA)—were also detected in rare cells in the stroma in one case, and in another case only one MA+ cell was detected. These results provide evidence for a lytic type of infection by EBV in the normal nasopharynx involving a very small proportion of stromal lymphocytes and support the view that nasopharyngeal lymphocytes can act as a reservoir for EBV in normal individuals. © 1995 Wiley-Liss, Inc.     

Inhibition of cell growth and Epstein-Barr virus reactivation by CD40 stimulation in Epstein-Barr virus-transformed B cells

The CD40 molecule plays important roles in B cell activation, proliferation, and immunoglobulin (Ig) class switching. In Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), CD40 mediates growth inhibition and EBV reactivation via the CD40 signaling pathways. CD40 cross-linking with a monoclonal antibody arrests cell growth in G1 and induces expression of p27kip1 cyclin-dependent kinase inhibitor. CD40 cross-linking also induces EBV reactivation, as detected by the induction of EBV-specific early antigen, immediate early BZLF1 RNA, and its protein product ZEBRA. These results support hypotheses that the proliferation of EBV-infected B cells in vivo can be inhibited by interactions with the CD40 ligand on activated helper T cells, and latent EBV is reactivated via the signaling pathways controlled by CD40 interactions.     

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

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

Expression of B-cell antigens by Hodgkin''s and Reed-Sternberg cells.

Twenty frozen and 55 paraffin sections of lymphnode specimens from 55 patients with pretreatment Hodgkin's disease (nodular sclerosis Hodgkin's disease, n = 45; mixed cellularity Hodgkin's disease, n = 10) were studied by immunohistochemistry and molecular analysis to determine the phenotype of Hodgkin's and Reed-Sternberg cells (HRS). In all cases the HRS cells were CD45-, and CD30+, and in 43/55 (78%) cases they were CD15+. In 48/55 cases (87%) HRS cells were reactive with at least one B-cell marker (CD19, CD20, CD22, CDw75, MB2), 8/55 cases (14.5%) showed reactivity (mainly cytoplasmic) of a subpopulation of HRS cells with the T-cell markers CD3 and beta F1. All cases that expressed T-cell antigens were also reactive with at least one B-cell marker. In frozen sections, a minority of HRS cells in each case studied showed cytoplasmic positivity for bcl-2 protein. Rearrangement of immunoglobulin heavy chain genes was detected in one case and of T-cell receptor beta chain genes in none. The authors were unable to confirm previous reports of bcl-2 gene rearrangement in Hodgkin's disease. The results strongly support a B lymphocytic origin of HRS cells.     

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen.

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.     

CD40 ligand inhibits Fas/CD95-mediated apoptosis of human blood-derived dendritic cells

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) in the immune system. In this study, we analyzed the regulation of apoptosis of human peripheral blood-derived DC. DC were generated from adherent peripheral blood mononuclear cells that had been cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. These cells displayed phenotypic properties of DC, including dendritic processes, expression of CD1a and lack of expression of CD14, and were very potent at presenting soluble antigens to T cells. Blood-derived DC were demonstrated to express the Fas/CD95 antigen and an agonist antibody to CD95 strongly induced apoptotic cell death in these cells. Soluble trimeric CD40 ligand potently inhibited both CD95-mediated and spontaneous apoptosis in DC. The data suggest that interactions between members of the tumor necrosis factor family of ligands expressed by T cells with their receptors on DC play an important role in the regulation of apoptosis in DC during antigen presentation and may, therefore, regulate the duration of T cell expansion and cytokine production.     

Epstein–Barr virus in Reed–Sternberg G-like cells in non Hodgkin's lymphomas

In the course of our study on Hodgkin's disease (HD), ten cases of non-Hodgkin's lymphomas (NHL) containing Hodgkin and Reed-Sternberg-like (MRS) cells were encountered. Many of these cases had initially been diagnosed as HD, but on careful review of the histology, with the aid of immunophenotyping studies, they were reclassified as NHL. The presence of Epstein–Barr virus (EBV) in these HRS-like cells was investigated using a combination of EBER in situ hybridization (ISH) and immunostaining for the detection of EBV-encoded latent membrane protein (LMP). HRS-like cells in four cases (two lymphoplasmacytoid lymphomas, one Richter's transformation of lymphoplasmacytoid lymphoma, and one immunoblastic lymphoma of T-cell type) were found to be EBV-positive. In two of these cases, a second biopsy taken up to 10 years later also contained EBV in the HRS-like cells. In three of the four cases, HRS-like cells expressed the activation antigen CD30, but the expression of B- or T-cell antigens was variable. All cases of T-cell-rich B-cell lymphomas were negative for EBV. In conclusion, EBV may play a role in the development of HRS-like cells i some cases of NHL. The relationship of HRS-like cells to HRS cells of HD is discussed.     

Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin''s disease.

The immunophenotype of the Reed-Sternberg cells in Hodgkin's disease is heterogeneous among different cases; this heterogeneity has contributed to the continuing uncertainty regarding the normal counterpart of the Reed-Sternberg cell. In this study, the authors demonstrate coexpression of the B-cell marker, CD20, and the granulocyte associated antigen, CD15, by Reed-Sternberg cells in three of 20 cases of nodular sclerosis and mixed cellularity Hodgkin's disease using a double-labelling technique in one case and staining of serial sections in three cases. Additionally, the authors found that expression of CD20 occurred more often in tumors with a monomorphous proliferation of mononuclear and binucleate Hodgkin's and Reed-Sternberg cells, without numerous eosinophils or polymorphonuclear neutrophils. In contrast, expression of CD15 by Reed-Sternberg cells was associated with a greater granulocyte infiltrate. The presence or absence of fibrosis, plasma cells, and histiocytes did not correlate with antigen expression. These results suggest that there may be a continuum of antigen expression by Reed-Sternberg cells, with some cells expressing CD20, some CD15, and others expressing both antigens; cells coexpressing both CD15 and CD20 may represent an unstable intermediate in the process of antigen switching. The possibility that antigen expression by the neoplastic cells in a given case may modulate depending on the background infiltrate could explain the heterogeneity of immunophenotype among cases of Hodgkin's disease.     

Down-regulation of Epstein-Barr virus nuclear antigen 1 in Reed-Sternberg cells of Hodgkin's disease.

AIMS--To demonstrate Epstein-Barr virus (EBV) encoded nuclear antigen 1 (EBNA-1) gene expression in EBV associated disorders using a new monoclonal antibody (1H4-1) on routinely processed tissues. METHODS--The pressure cooker antigen retrieval method was used for the immunohistochemical demonstration of EBNA-1 gene expression in formalin fixed, EBV positive tissues from Hodgkin's disease, infectious mononucleosis, HIV associated non-Hodgkin's lymphomas, post-transplant lymphomas, and undifferentiated nasopharyngeal carcinoma (NPC). EBV encoded EBNA-2, latent membrane protein 1 (LMP-1) and BZLF-1 gene expression was also examined using commercially available monoclonal antibodies. RESULTS--Of the 34 EBER in situ hybridisation positive cases of Hodgkin's disease examined, none expressed EBNA-1 in the Reed-Sternberg cells. These cells were nevertheless strongly LMP-1 positive in all cases. Strong EBNA-1 staining was seen in all cases of EBER positive HIV associated non-Hodgkin's lymphoma (five of five), nasopharyngeal carcinoma (five of five), infectious mononucleosis (three of three), and post-transplant lymphoma (one of one). These cases also expressed LMP-1, EBNA-2 and BZLF-1, but at differing levels. CONCLUSION--The pressure cooker antigen retrieval procedure is a sensitive and reliable adjunct to immunohistochemistry, especially with antibodies which are otherwise ineffective on routinely processed tissues. The EBNA-1 gene is not expressed at detectable levels in the malignant cells of Hodgkin's disease, but is consistently expressed in other EBV associated disorders. This finding has important implications for the role of EBNA-1 in the biology of EBV.     

Decreased expression of the ligand for CD40 in newborn lymphocytes

Immune responses in newborn lymphocytes show a defect in isotype switching from IgM to IgG and IgA. Immunoglobulin isotype switching in B lymphocytes requires a contact-dependent signal from T lymphocytes which is delivered by the ligand for the B cell surface antigen CD40. We investigated the capacity of newborn lymphocytes to express the CD40 ligand and to undergo CD40 ligand-dependent immunoglobulin isotype switching. After stimulation by phorbol ester and ionomycin, newborn lymphocytes expressed markedly decreased amounts of CD40 ligand on their surface compared to normal adult lymphocytes. Northern blot analysis of mRNA derived from activated cord blood lymphocytes also revealed markedly decreased amounts of CD40 ligand mRNA. Decreased expression of CD40 ligand in newborn lymphocytes was associated with a severely decreased ability to undergo T cell-dependent immunoglobulin isotype switching. Newborn lymphocytes synthesized little or no detectable IgE in response to T cell-dependent stimulation by interleukin-4 but synthesized IgE in response to T cell-independent stimulation by CD40 monoclonal antibody and interleukin-4. These results indicate that decreased expression of CD40 ligand in newborn lymphocytes may be the underlying cause of deficient immunoglobulin isotype switching in newborns.     

A novel immunotherapy to hepatocellular carcinoma: CD40-activated B lymphocytes transfected with AFPmRNA

Alpha-fetoprotein (AFP) is overexpressed in the majority of hepatocellular carcinomas (HCCs), and thus may offer attractive target for immunotherapy against this neoplasm. CD40 ligand (CD40L) is the major signal that induces B cells to efficiently present antigen to T cells, and CD40-activated B (CD40-B) lymphocyte cells may boost cytotoxic T lymphocytes (CTLs) when they are pulsed with tumour antigens. CTL is considered to be a promising therapeutic means for the treatment of cancers. Here, we intend to build a plasmid pGEM4Z/AFP/A64 and to prepare AFPmRNA, then separate B lymphocyte cells. These CD40-B cells are pulsed with AFPmRNA, and they may boost robust T-cell responses, but more importantly, they also may prime naive T-cell responses against hepatocarcinoma. These CD40-B cells will be a powerful source of APCs generated by simple and reliable technology that may be applied to antigen responses, immune treatment for cancer, vaccination approaches, and ex vivo T-cell expansion for adoptive therapy. AFPmRNA-transfected B cells may represent a broadly applicable vaccine strategy to induce potentially therapeutic CTL responses against AFP-positive target cells in HCC. Vaccine strategies such as these may contribute to the effective future treatment of HCC.     

Presentation of epstein-barr virus latency antigens to CD8(+), interferon-gamma-secreting, T lymphocytes

Epstein-Barr virus (EBV) infects more than 95 % of the human population and causes an asymptomatic life-long infection in the majority of EBV carriers. Cell-mediated immunity provides resistance to EBV, as demonstrated by the occurrence of EBV-induced post-transplant lymphoproliferative disease in immunosuppressed patients. Here we looked for IFN-gamma-producing T lymphocytes in the blood of healthy donors with a rapid enzyme-linked immunospot (ELISPOT) assay, comparing as antigen presenting cells monocytes and dendritic cells (DC) infected with recombinant vaccinia virus (rVV). We found a strong CD8(+) ELISPOT response to one or more of the EBNA 3A, 3B and 3C antigens in the PBMC from 14 / 18 donors. The sensitivity of the overnight ELISPOT assay was increased using DC as antigen-presenting cells, including 3 / 3 individuals who lacked ELISPOT in PBMC. In addition, DC could markedly expand EBV-specific spots after a 7-day culture. In a smaller number of donors, we documented recognition of the subdominant LMP 1, LMP 2 and EBNA 1 antigens that are expressed in a variety of EBV-associated malignancies. Therefore our data provide more evidence for the efficacy of DC in eliciting rapid responses to EBV latency antigens in circulating CD8(+) T cells.     

CD40-CD40 ligand interactions stimulate B cell antigen processing

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II E^k molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.