Selectins (CD62L, CD62P) and megakaryocytic glycoproteins (CD41a, CD42b) mediate megakaryocyte-fibroblast interactions in human bone marrow

Previous in vitro studies are in keeping with the finding that isolated and enriched megakaryocytes attach to bone marrow fibroblasts and generate an increased growth of these cells. This process was assumed to depend on a close spatial relationship between both cell types which supports the paracrine effect of platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1. Moreover, adhesion molecules including β1 integrin receptors and fucosylated structures were determined to play an important role in these complex interactions. However, up to now the influence of megakaryocyte expressed glycoproteins CD41a and CD42b in these processes was not investigated. In addition, the role of megakaryocytic CD62P and also of CD62L, both adhesion molecules of the selectin group, could also be of interest. Following isolation and enrichment of bone marrow megakaryocytes and fibroblasts, both cell populations were characterized regarding their expression of these factors by applying immunocytochemical techniques. Additionally, their influence on adhesion of megakaryocytes to fibroblasts as well as fibroblast growth was evaluated by comparative megakaryocyte–fibroblast co-cultures and inhibition studies using specific monoclonal antibodies (mabs). Fibroblast monocultures served as controls. In these experiments, selectin-specific antibodies significantly reduced megakaryocyte attachment to fibroblast feeder layers and fibroblast growth in the co-cultures. The effect of CD41a and CD42b specific antibodies was limited to megakaryocyte-dependent fibroblast growth. These results elucidate the involvement of the selectins CD62P and CD62L in the basal activation of megakaryocytes inducing their attachment to bone marrow fibroblasts. In contrast, the megakaryocyte glycoproteins CD41a and CD42b exert their effect on the megakaryocyte dependent fibroblast growth. Altogether, it is tempting to speculate that the various interactions of these mediators reflect certain steps in the complex pathomechanisms causing the evolution of (reactive) myelofibrosis in hematopoietic neoplasias accompanied by megakaryocytic proliferation.     

Factors Associated with Dysplastic Changes in Sinonasal Inverted Papilloma: Study of Tumor Infiltrating Lymphocytes (TILs) FOXP3, CD4, CD8, and expression of p53

Objective: This study examine FOXP3, CD4, CD8 and p53 expression in the transformation of the Sinonasal Inverted Papilloma (SIP) malignancy into sinonasal carcinoma. Materials and Methods: This study used a cross-sectional approach. The research sample from thirty-six paraffin block preparations with the diagnosis of SIP. Then, immunohistochemical staining was performed using FOXP3 mouse monoclonal antibody (236A/E7), CD8 rabbit monoclonal antibody (CD8/1179R), CD4 mouse monoclonal antibody (4B12) and p53 rabbit monoclonal antibody.  Results: There was a significant difference between Foxp3 expression in SIP without dysplasia and SIP with dysplasia (p= 0.013). There was no significant difference between the expression of CD4 and CD8 in the two groups with p-values 0.1 and 0.062, respectively. The mean percentage of positive p53 expression in SIP without dysplasia was 0.45+0.63 and in the SIP with dysplasia 29.31+38.96. There was a significant difference between the two groups (p<0.001). Conclusion: FOXP3 and p53 were overexpressed in SIP with malignant transformation. FOXP3 together with p53 status is associated with dysplastic changed in the SIP. FOXP3 and p53 status could be potential biomarker of malignant transformation in sinonasal inverted papilloma.     

Expression and distribution of S-100, CD83, and costimulatory molecules (CD80 and CD86) in tissues of thyroid papillary carcinoma

A higher expression of S-100 in tissue of thyroid papillary carcinoma (TPC) vs. thyroid follicular adenoma (TFA) (p < .001) was observed as well as a higher expression of CD83 in the peri-cancerous tissues vs. TFA (p < .001), oppositely, CD83 was negative in the cancerous net. TPC showed greater decreases in levels of CD80 and CD86 than did the TFA. These findings suggest that impaired immune function, absence of CD83-positive mature and activated dendritic cells in cancer nodules may have a role in the pathogenesis of TPC. The low expression of CD80 and CD86 in TPC may help them evade the immune system.     

Ecto-5'-Nucleotidase (E5'-NT, 27.2, CD73), 27.1, Leu13, CD28 and LAM-1(Leu8) Antigens in Mycosis Fungoides (MF)

Two monoclonal antibodies (MoAbs) B121 and B124 to the human lymphocyte differentiation antigen E5'-NT (CD73), and the MoAbs 27.2 and 27.1 that were raised to HTLV-1⁺CD4⁺CD25⁺ leukemic T cells were tested immunohistochemically on frozen sections of 13 cases of MF together with Leu13, CD28, LAM-1(Leu8) and other standard T-cell markers. Controls included 7(5T, 2B) non-MF cutaneous non-Hodgkin's lymphomas (NHL), 2 cutaneous T lymphoid leukemias (T-ALL), 13 miscellaneous non-neoplastic dermatoses, 11(5T, 6B) extracutaneous NHL, and 5 splenic B-hairy cell leukemias. Previous studies suggest that 27.2 also recognizes the CD73 antigen and is present in high density in some cases of HTLV-1⁺ adult thymic leukemia/lymphoma (ATL) and HTLV-1^- Sezary syndrome (Y. Fukunaga et al., Blood 74:2486-2492, 1989). In the studies reported here B121, B124 and 27.2 reacted similarly with the MF and control samples tested. The CD73 antigen was present in the majority of lymphoid cells from 8 (62%) of 13 cases of MF. In one of these cases the CD73 antigen was detected only in the deeper and more pleomorphic MF dermal infiltrate. Although CD73 expression was noted in varying numbers of lymphocytes from many of the control benign and malignant lymphoproliferative lesions of the skin, none of the extracutaneous NHL or HCL cases tested showed significant numbers of CD73⁺ cells. Ten, 11 and 11 of the 13 MF cases also showed moderate to high numbers of cells reactive with 27.1, Leu13 and CD28, respectively. Most of the MF cases also showed moderate to high co-expression of βF1 (TCRβ), CD3 and CD4, and 9 cases showed moderate numbers of CD8+ cells as well. In contrast to the control dermatoses, where significant co-expression of the majority T-cell markers LAM-1(Leu8), CD5 and CD7 was the rule, 11 of the 13 MF cases showed significant loss of one or more of these antigens. One or more of these antigens was notably also lost with CD73 in 3 patients who had, or developed, systemic MF. The above results indicate that MF cases often co-express the CD73/27.2 antigen with 27.1, Leu13, CD28, βF1 (TCRβ), CD3 and CD4, and may show a concurrent absence of CD5, CD7 or LAM-1(Leu8) antigens with or without loss of CD73. The possible roles of CD73, Leu 13, CD28 and LAM-1(Leu8) in controlling cell proliferation, adhesion and migration in MF are discussed.     

Prominin-1 (CD133, AC133) and dipeptidyl-peptidase IV (CD26) are indicators of infinitive growth in colon cancer cells

Advanced colorectal cancer is characterized by uncontrolled growth and resistance against anti-cancer agents, including ErbB inhibitors. Recent data suggest that cancer stem cells (CSC) are particularly resistant. These cells may reside within a CD133+ fraction of the malignant cells. Using HCT116 cells we explored the role of CD133 and other CSC markers in drug resistance in colon cancer cells. CD133+ cells outnumbered CD133- cells over time in long-term culture. Both populations displayed the KRAS mutation 38G > A and an almost identical target profile, including EGFR/ErbB1, ErbB2, and ErbB4. Microarray analyses and flow cytometry identified CD26 as additional CSC marker co-expressed on CD133+ cells. However, knock-down of CD133 or CD26 did not affect short-term growth of HCT116 cells, and both cell-populations were equally resistant to various targeted drugs except irreversible ErbB inhibitors, which blocked growth and ERK1/2 phosphorylation in CD133- cells more efficiently than in CD133+ cells. Moreover, the MEK inhibitor AS703026 was found to overcome resistance against ErbB blockers in CD133+ cells. Together, CD133 and CD26 are markers of long-term growth and resistance to ErbB blockers in HCT116 cells, which may be mediated by constitutive ERK activity.     

Lipoplex mediated silencing of membrane regulators (CD46, CD55 and CD59) enhances complement-dependent anti-tumor activity of trastuzumab and pertuzumab

The therapeutic potential of anticancer antibodies is limited by the resistance of tumor cells to complement-mediated attack, primarily through the over-expression of membrane complement regulatory proteins (mCRPs: CD46, CD55 and CD59). Trastuzumab, an anti- HER2 monoclonal antibody, approved for the treatment of HER2-positive breast and gastric cancers, exerts only minor complement-mediated cytotoxicity (CDC). Pertuzumab is a novel anti-HER2 monoclonal antibody, which blocks HER2 dimerization with other ligand-activated HER family members. Here, we explored the complement-mediated anti-tumor effects of trastuzumab and pertuzumab on HER2-positive tumor cells of various histological origins.Delivery of chemically stabilized anti-mCRP siRNAs using cationic lipoplexes, AtuPLEXes, to HER2-over-expressing BT474, SK-BR-3 (breast), SKOV3 (ovarian) and Calu-3 (lung) cancer cells reduced mCRPs expression by 85–95%. Knockdown of individual complement regulators variably led to increased CDC only upon combined treatment with trastuzumab and pertuzumab. The combined down-regulation of all the three regulators augmented CDC by 48% in BT474, 46% in SK-BR-3 cells, 78% in SKOV3 cells and by 30% in Calu-3 cells and also increased complement-induced apoptosis and caspase activity on mCRP neutralized tumor cells. In addition, antibody-induced C3 opsonization of tumor cells was significantly enhanced after mCRP silencing and further augmented tumor cell killing by macrophages.Our findings suggest that siRNA-induced inhibition of complement regulator expression clearly enhances complement- and macrophage-mediated anti-tumor activity of trastuzumab and pertuzumab on HER2-positive tumor cells. Thus – if selectively targeted to the tumor – siRNA-induced inhibition of complement regulation may serve as an innovative strategy to potentiate the efficacy of antibody-based immunotherapy.     

Expansion of large granular lymphocytes (natural killer cells) with limited antigen expression (CD2+, CD3-, CD4-, CD8-, CD16+, NKH-1-) in a human immunodeficiency virus-positive homosexual man

Lymphoid neoplasms associated with acquired immune deficiency syndrome (AIDS) are mostly of B-cell type and rarely of T-cell origin. The authors report a case of a homosexual HIV antibody-positive, HTLV-1 antibody-negative man who developed T-lymphoproliferative disorder (TGLD) after he experienced a viral-like illness. The lymphoproliferative disorder was characterized by increased peripheral blood large granular lymphocytes (LGL) with azurophilic granules (natural killer [NK] cells) which had limited antigen expression: CD2+, CD3-, CD4-, CD8-, CD16+, NKH-1-. The LGL failed to express T-cell or T-cell-related antigens, with the exception of CD2. No functional or gene rearrangement studies were performed on the patient's lymphocytes. However, the results of immunophenotyping, including CD25, W26, and HLA-DR, were suggestive of an inactive state, and the negative finding for CD3 antigen was consistent with unarranged gene T-cell receptors. This is the first reported case of TGLD in an HIV antibody-positive patient.     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: II, Aberrant HLA-ABC Expression by CD45RA and CD45RO Cell Subpopulations of Mature CD4+ T-Cell Leukaemias

Six thymocyte suspensions, 10 normal blood CD4⁺ CD8^- lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4⁺ mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4⁺ CD8^- blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a ⁺ 2H4^- UCHL1⁺ (25 HLA-ABC fluorescent units), (b)CD1a^-2H4^intUCHL1⁺ (134 units), and (c) CD 1a^- 2H4 ⁺ UCHL1 ^- (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4⁺ 2H4⁺ (318 units) and CD4 ⁺ 2H4^- (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4⁺ and 2H4^- components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a^- thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4⁺ 2H4⁺ (710 units) and CD4⁺ 2H4^- (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 ⁺ components, with only 14% of 2H4^- fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4^-UCHL1⁺ subpopulation in CD4⁺ malignancies had a lower HLA-ABC level than the 2H4⁺UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.     

Programmed cell death: the influence of CD40, CD95 (Fas or Apo-I) and their ligands

Programmed cell death (PCD) or apoptosis is a process whereby developmental or environmental stimuli activate a specific series of events that culminate in cell death. PCD is essential for normal development and abnormality in the process can lead to defects ranging from embryonic lethality and tissue-specific perturbation of postnatal development to a high susceptibility to malignancy. Therapeutics that modulate the regulation of PCD may provide a new opportunity for the treatment of the PCD related diseases and cancer. CD40 and CD95 (Fas/Apo-I) are transmembrane proteins of the nerve growth factor/tumour necrosis factor α receptor superfamily. The death signal of PCD occurs when the CD95 receptor on the cell surface binds to the CD95 ligand (CD95L) or to the anti-CD95 monoclonal antibody (mAb). In contrast, PCD could be inhibited by the survival signal mediated from the binding of the CD40 receptor to the CD40 ligand (CD40L) or to the anti-CD40 mAb. In this review, the interaction of CD40/CD40L and CD95/CD95L on PCD in normal and malignant cells is discussed.     

Expression of c-kit Receptor (CD 117) and CD34 in Leukemic Cells

The expression of c-kit receptor (c-kit R; CD117) and CD34 was examined in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) in blastic transformation (BT), and myelofibrosis (MF) in myeloid BT. In myeloid leukemia including AML, CML-myeloid BT and MF-myeloid BT, both c-kit R and CD34 were expressed synchronously, while in lymphoid leukemia including ALL and CML-lymphoid BT, only CD34 was highly expressed. A close correlation between c-kit R and CD33 expression and an inverse correlation between c-kit R and CD 19 expression were observed when all of the myeloid plus lymphoid leukemia cells were analysed. There was a close correlation between c-kit R and CD34 expression in the myeloid leukemia cells, c-kit R expression may be associated with myeloid phenotypes of leukemic cells and may be useful for the diagnosis of myeloid leukemia. The literature of c-kit R expression in leukemic cells is reviewed here and the comparison of c-kit R and CD34 expression in normal hematopoietic progenitor cells with those on the leukemic counterparts was discussed.     

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: I, Diverse Patterns of Expression in Mature (Post-Thymic) T-Cell Proliferations

By simultaneous two- and three-colour flow cytometry, this study analysed the expression of membrane CD45RA (2H4) and CD45RO (UCHL1) determinants by normal thymocytes (n = 5) and peripheral blood lymphocyte subpopulations (CD4⁺, n = 21; CD8⁺, n = 12; CD8^dim+, n = 12) and compared these patterns with those of T-cells from representative CD4⁺CD8^- (n = 8), CD4⁺CD8⁺ (n = 2), CD4^-CD8⁺ (n = 10) and CD4^-CD8^- (n = 1) proliferations. These comprised cases of prolymphocytic leukaemia (T-PLL, n = 5), adult T-cell leukaemia-lymphoma (ATLL, n = 2), Sezary Syndrome (SS, n = 4), chronic lymphocytic leukaemia (T-CLL, n = 4), and lymphoproliferative disease of granular lymphocytes (LDGL, n = 5). Normal thymocyte fractions, of which a mean of 85% cells co-expressed membrane CD4 and CD8, were predominantly (mean 89%) 2H4^-UCHL1⁺ with the remaining cells consisting of 2H4^intUCHL1⁺ and 2H4⁺UCHL1^- components. Further analysis showed that virtually all CDla⁺ thymocytes were UCHL1⁺ whereas the CD1a^- fraction comprised similar proportions of both UCHL1^- and UCHL1⁺ subpopulations. Similarly, normal blood CD4⁺, CD8⁺ and CD8^dim+ lymphocytes showed reciprocal CD45RA/CD45RO expression and could be phenotypically grouped into 2H4⁺UCHL1^- 2H4^intUCHL1⁺ and 2H4^-UCHL1⁺ subpopulations. Mean proportions of 48% and 68%, for CD4⁺ and CD8⁺ lymphocytes respectively, showed a composite 2H4⁺UCHL1^- phenotype, whereas the percentage of NK-associated CD8^dim+ cells with this phenotypic pattern was considerably higher (mean, 85%). Normal lymphocyte subpopulations lacking both determinants (2H4^-UCHL1^-) were only rarely noted. Comparing normal patterns of CD45RA/CD45RO expression with those of the T-cell proliferations revealed diverse and abnormal patterns of staining for 3/6 of the CD4⁺CD8^- SS and ATLL, and for 5/5 of the T-PLL (CD4⁺CD8^-, n = 2; CD4⁺CD8⁺, n = 2; and CD4^-CD8⁺, n = 1) cases studied. In contrast, the nine cases of CD4^-CD8⁺ T-CLL and LDGL all showed CD45RA/CD45RO staining patterns similar to that of normal CD8⁺/CD8^dim+ blood lymphocytes (i.e. a predominance of 2H4⁺UCHL1^- cells). Although the variant CD45RA/CD45RO pattern types of the CD4⁺ proliferations did not appear to be related to either the diagnostic category or other phenotypic characteristics, the high proportion of abnormal patterns within this case group suggests that recognition of these abnormalities may be potentially relevant to the differentiation of benign and malignant CD4⁺ proliferations and, in addition, may be of aetiological importance with respect to the diverse acquired defects in immunity commonly seen in patients with such disorders.     

Isolation of CD24(high) and CD24(low/-) cells from MCF-7: CD24 expression is positively related with proliferation, adhesion and invasion in MCF-7

CD24 has been reported to have a role in metastasis of several malignancies including breast cancer. We investigated the effect of CD24 expression on biologic features in human breast cancer cells, and found that CD24 expression is associated with more rapid growth and enhanced ability of adhesion and invasion in MCF-7. The proportion of cells was higher in synthetic phase and lower in arrestic phase for CD24(high) than for CD24(low/-) cells. Cyclin D1 and p27 had stronger expressions in CD24(low/-) cells than CD24(high) cells. These suggested one possible pathway for CD24 effect on proliferation.     

Decay-accelerating factor (DAF, CD55) in normal colorectal mucosa, adenomas and carcinomas.

Decay-accelerating-factor (DAF, CD55), a phosphatidyl-inositol anchored glycoprotein, is a member of the cell membrane bound complement regulatory proteins that inhibit autologous complement cascade activation. DAF was found expressed on cells that are in close contact with serum complement proteins, but also on cells outside the vascular space and on tumour cells. Using CD55(BRIC110) and CD55(143-30) we show here that DAF(CD55) is only sporadically expressed on the luminal surface of normal colonic epithelium. However, 5/20 adenomas expressed DAF(CD55) on the cell surface of all tumour cells, 5/20 adenomas were completely negative, 10/20 adenomas expressed DAF(CD55) in various amounts. DAF(CD55) was expressed in various intensities on almost all tumour cells of the colon carcinoma cell line HT29. In 5/88 colorectal carcinomas DAF(CD55) was localised on the apical cell surface of all tumour cells, 31/88 were completely negative, 52/88 expressed DAF(CD55) in parts of their neoplastic populations. There was no correlation between the tumour grading, staging and location and the mode of DAF(CD55) expression, but DAF(CD55) was found more often in mucinous carcinomas (P = 0.007). Although the mode of DAF(CD55) expression is not correlated with tumour prognostic parameters, the upregulation of DAF(CD55) in a subset of adenomas and carcinomas needs further investigation concerning protection of tumour cells against complement cytotoxicity.     

Enhancement of polymorphonuclear cell-mediated tumor cell killing on simultaneous engagement of fcgammaRI (CD64) and fcalphaRI (CD89)

Antibodies can efficiently induce antitumor responses via recruitment of Fc receptor-bearing cytotoxic cells. Polymorphonuclear (PMN) cells represent attractive effector cells for antibody-directed immunotherapy. This, because activated PMN cells coexpress the class I receptors for IgG (FcgammaRI, CD64) and IgA (FcalphaRI, CD89), which are potent cytotoxic trigger molecules. Both receptors, however, require the FcR gamma chain for signaling. In this study, we show that FcgammaRI and FcalphaRI can trigger function independently of one another and do not cross-compete for the FcR gamma chain. FcalphaRI proved more efficient in initiating early signaling events and effector functions, such as redirected tumor cell killing and generation of superoxide. In addition, simultaneous engagement of FcgammaRI and FcalphaRI resulted in enhanced tumor cell lysis. These data support the development of concepts in which both FcgammaRI and FcalphaRI on PMN cells are targeted for tumor therapy.     

Abundant expression of CD40 and CD40-ligand (CD154) in paediatric Langerhans cell histiocytosis lesions

The pathogenesis of Langerhans cell histiocytosis (LCH) is obscure, partly because the events leading to activation of Langerhans-like lesional cells (LCH cells) and associated T cells, and the excessive cytokine production by these cells are unknown. The interaction between CD40 on antigen-presenting cells (APC) like Langerhans cells and CD40 ligand (CD40L) (CD154) expressed by activated CD4+ T cells, is essential for the activation of both the APC and the T cells and results in upregulation of APC functions and initiation of immunoreactivity. The effects of CD40-CD40L interaction include increased expression of co-stimulatory and adhesion molecules, proliferation, and production of pro-inflammatory cytokines and proteolytic enzymes, all features of LCH. Using immunohistochemistry, we analysed the in situ presence of the co-stimulatory molecules CD40 and CD40L in 15 fresh frozen biopsies of LCH lesions in children. The cells producing these molecules were identified by double staining for CD1a on LCH cells and CD3 on T cells. Prominent expression of CD40 by LCH cells and CD40L by T cells was found in all 15 specimens regardless of the source of specimen or characteristics of the patient. The findings of high expression of CD40 and CD40L in all specimens imply a key role for the CD40-CD40L adhesion pathway in the pathogenesis of LCH. Since this interaction is an accessible and realistic target for immunotherapy, these findings prompt speculation on the use of blocking antibodies to CD40 or to CD40L in the treatment of LCH.     

CD40-expressing carcinoma cells induce down-regulation of CD40 ligand (CD154) and impair T-cell functions

The interaction of CD40 expressed by immunocompetent cells with its ligand CD154 on the surface of T-helper cells plays a crucial role in the immune response. Recently, the presence of CD40 was also demonstrated on a variety of carcinomas. Whereas the critical relevance of CD40 in cytotoxic T-cell priming via dendritic cells is already established, the biological role of CD40/CD154 interactions in nonhematopoetic cells is still unclear. In the present study we demonstrate that CD154 expression density is down-regulated on activated T cells on interaction with CD40+ tumor cells. Naive T cells cocultured with CD40+carcinoma showed impaired functionality as indicated by a reduced frequency of IFN-gamma secreting cells, reduced interleukin 2 secretion, impaired proliferation, and a lack of CD154 re-expression on restimulation. In distinction, T-cell effector lysing capacity was not impaired by CD40-expressing tumor cell targets. The present results suggest that in marked contrast to antigen-presenting cells, CD40 expression on carcinoma cells suppresses T-cell activation. Our findings support the statement that CD40 functions are context dependent and imply a new function for CD40 expressed on nonantigen-presenting cells.     

Ex vivo pediatric brain tumors express Fas (CD95) and FasL (CD95L) and are resistant to apoptosis induction

Fas (APO-1/CD95/TNFRSF6) is a member of the tumor necrosis/nerve growth factor receptor family that signals apoptotic cell death in sensitive cells. Expression of Fas and its agonistic ligand (FasL/TNFSF6) was investigated in ex vivo pediatric brain tumor specimens of various histologic types. Fas expression was identified in all of the 18 tumors analyzed by flow cytometry and immunohistochemistry. FasL expression was identified in most of the 13 tumors analyzed by both Western analysis and immunohistochemistry. Nine of these tumor specimens were treated with either the agonistic anti-Fas antibody (APO-1) in combination with protein A or FasL in short-term cytotoxicity assays. Sensitivity to apoptosis induced by the topoisomerase II inhibitor, etoposide, was also assessed. Despite the presence of Fas, all the specimens analyzed demonstrated a high degree of resistance to Fas-mediated apoptosis. These 9 specimens also showed a high degree of resistance to etoposide. Only 2 of the 9 specimens were susceptible to etoposide-induced cell death, whereas only 3 were sensitive to Fas-mediated apoptosis. One brain tumor was sensitive to both Fas ligation and etoposide treatment. This contrasted with the high degree of susceptibility to both etoposide- and Fas-induced apoptosis observed in the reference Jurkat cell line. The results suggest that Fas expression may be a general feature of tumors of the CNS and that a significant degree of resistance to Fas-mediated apoptosis may exist in ex vivo pediatric brain tumor specimens.