Danny F. Dukers

Direct Immunosuppressive Effects of EBV-Encoded Latent Membrane Protein 1

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-γ release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-γ release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.

Unique polycomb gene expression pattern in Hodgkin's lymphoma and Hodgkin's lymphoma-derived cell lines

Human Polycomb-group (PcG) genes play a crucial role in the regulation of embryonic development and regulation of the cell cycle and hematopoiesis. PcG genes encode proteins that form two distinct PcG complexes, involved in maintenance of cell identity and gene silencing patterns. We recently showed that expression of the BMI-1 and EZH2 PcG genes is separated during normal B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS) cells co-express BMI-1 and EZH2. In the current study, we used immunohistochemistry and immunofluorescence to determine whether the binding partners of these PcG proteins are also present in H/RS cells and H/RS-derived cell lines. PcG expression profiles were analyzed in combination with expression of the cell cycle inhibitor p16INK4a, because experimental model systems indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and HL-derived cell lines co-express all core proteins of the two known PcG complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1, and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in normal mature B cells and other malignant lymphomas of B-cell origin, suggesting that the PcG expression profile of H/RS is unique. In contrast to Bmi-1 transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL revealed strong nuclear expression of p16INK4a. We propose that abnormal expression of BMI-1 and its binding partners in H/RS cells contributes to development of HL. However, abnormal expression of BMI-1 in HL is not necessarily associated with down-regulation of p16INK4a.

Quantitative immunohistochemical analysis of cytokine profiles in Epstein-Barr virus-positive and -negative cases of Hodgkin's disease

Hodgkins disease (HD) is a malignant lymphoproliferative disease characterized by the presence of Hodgkin–Reed–Sternberg cells surrounded by a reactive infiltrate. In Epstein–Barr virus (EBV)‐associated cases (40–60%), at least two EBV‐encoded proteins [latent membrane protein 1 (LMP1) and LMP2] are expressed, which are potential targets for cytotoxic T‐lymphocytes (CTLs). Although in EBV‐positive cases significantly more activated (granzyme B‐positive) CTLs and natural killer (NK) cells are present, the cytotoxic immune response is not sufficient for adequate killing of tumour cells. The production of immunomodulating cytokines within the tumour may be one of the mechanisms causing circumvention of the immune system. This study investigated by immunohistochemistry the presence of the immunosuppressive cytokine interleukin‐10 (IL‐10) and other Th1/Th2‐associated cytokines [IL‐2, IL‐4, interferon‐gamma (IFN‐γ)] in the neoplastic cells and reactive lymphocytes of nine EBV‐positive and 18 EBV‐negative cases of HD. The percentage of IL‐10‐expressing cells, both neoplastic and reactive, in EBV‐positive cases was significantly higher (33.1% vs. 18.5% for the neoplastic cells and 21.6% and 12.2% for the reactive cells, p=0.003 and 0.04, respectively) than in EBV‐negative cases. No difference in the percentage of IL‐2‐, IL‐4‐ and IFN‐γ‐expressing cells was observed. These results suggest that escape from local immune surveillance is not due to a shift from Th1 towards Th2, but may be caused by a direct effect of IL‐10 on the cytotoxic cells. Copyright

CD8+ T Cells in Cutaneous T-Cell Lymphoma: Expression of Cytotoxic Proteins, Fas Ligand, and Killing Inhibitory Receptors and Their Relationship With Clinical Behavior

PURPOSE We investigated the number, phenotype, and prognostic significance of CD8+ T cells in patients with mycosis fungoides (MF) and CD30- primary cutaneous large T-cell lymphoma (PCLTCL). PATIENTS AND METHODS Immunohistochemical stainings for CD8, granzyme B (GrB), T cell-restricted intracellular antigen (TIA-1), Fas ligand (FasL), and killer-cell inhibitory receptors (KIRs; CD95, CD158a, and CD158b) were performed on 83 first-diagnostic biopsy samples obtained from patients with plaque-stage MF (n = 42), tumor-stage MF (n = 20), and CD30- PCLTCL (n = 21). RESULTS Serial sections and double-staining experiments showed that the large majority of CD8+ T cells in MF and CD30- PCLTCL expressed TIA-1 and FasL, whereas only a minority expressed GrB, which suggested that these CD8+ T cells were partly activated cytotoxic T lymphocytes (CTLs). These CD8+ CTLs never or rarely expressed KIRs. This phenotype was a constant feature of CD8+ CTLs and did not alter with disease progression. In contrast, the median percentage of CD8+ CTLs in plaque-stage MF (22%), tumor-stage MF (7%), and CD30- PCLTCL (3%) differed significantly (P < .0001) and was associated with a significant decrease in 5-year survival. Also within the group of tumor-stage MF, a significant relation between CD8+ CTLs and survival was found. Multivariate analysis in the total group of MF demonstrated that both skin stage and percentage of CD8+ CTLs were independent parameters of survival. CONCLUSION Our results demonstrated that partly activated CD8+ CTLs were present in CTCL and that high proportions of these cells correlated with a better prognosis. This suggested that these CD8+ CTLs could play an important role in the antitumor response in these conditions.

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients

BACKGROUND In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.

High numbers of granzyme B/CD8-positive tumour-infiltrating lymphocytes in nasopharyngeal carcinoma biopsies predict rapid fatal outcome in patients treated with curative intent.

This study determined whether tumour‐infiltrating lymphocytes (TILs) in nasopharyngeal carcinomas (NPCs) include activated cytotoxic T lymphocytes (CTLs) and whether the numbers of activated CTLs in these biopsies are related to clinical outcome. Moreover, the study investigated whether the numbers of activated CTLs are associated with the expression of MHC class I proteins and the granzyme B antagonist PI‐9 in the tumour cells. Forty‐three Indonesian NPC patients (T1–3, N1–3, M0), who were treated with curative intent by radiotherapy only, were studied. Tumour‐infiltrating activated CTLs were detected using antibodies against granzyme B, CD8, and CD56. Expression of MHC class I proteins and PI‐9 was also determined by immunohistochemistry. Granzyme B‐positive TILs were detected in all NPC biopsies. The presence of a high percentage (>25%) of granzyme B‐positive TILs appeared to be a very strong predictor of a rapid fatal clinical outcome, independent of stage. Complete absence of MHC class I heavy chain expression in tumour cells was observed in 11 of 31 evaluable cases and low levels were observed in seven additional cases. No association between MHC class I expression and the numbers of granzyme B‐positive TILs was observed. Expression of the granzyme B antagonist PI‐9 in tumour cells was detected in three cases. It is concluded that the presence of many granzyme B‐positive TILs in a selected group of Indonesian NPC patients is a strong and stage‐independent marker for a rapid fatal clinical outcome. Copyright

Rb, mcl-1 and p53 expression correlate with clinical outcome in patients with liver metastases from colorectal cancer

BACKGROUND Thymidylate synthase (TS) has been associated with clinical outcome in disseminated colorectal cancer. However, many patients with low TS expression still fail to respond to treatment. Therefore, we studied the cell cycle proteins, Rb, E2F2, Ki67, p21 and p53 and the apoptotic proteins, mcl-1, hax, bcl-xl, bcl-2, Fas receptor, Fas ligand, caspase-3, M30 and PARP as potential predictive factors. PATIENTS AND METHODS In biopsy specimens of liver metastases from 31 colorectal cancer patients, protein expression was retrospectively determined by immunohistochemistry and related to response to hepatic arterial or intravenous (i.v.) 5-fluorouracil (5-FU) treatment, time to tumour progression (TTP) and overall survival. RESULTS Expression of both p53 and Rb correlated with survival benefit after 5-FU treatment. A median survival time of 79 weeks was found in patients with high levels of p53 or Rb compared to 36 and 44 weeks for patients expressing low levels of p53 (P = 0.027) or Rb (P = 0.030), respectively. Multivariate analysis showed that p53 was the best predictor of survival independent of sex, age or prior treatment. Following 5-FU hepatic arterial infusion, patients with a high TS expression had a shorter survival time than those with a low expression (P = 0.025). The anti-apoptotic protein mcl-1 was the only factor, which correlated with response to 5-FU treatment. Thirty-five percent of patients with a diffuse mcl-1 expression responded whereas ninety percent of patients with a peri-nuclear expression responded (P = 0.041). CONCLUSIONS These results indicate that besides TS, also Rb, p53 and mcl-1 are correlated with clinical outcome in patients with liver metastases from colorectal cancer.

Distinct expression patterns of polycomb oncoproteins and their binding partners during the germinal center reaction

Polycomb group (PcG) genes encode two chromatin‐binding protein complexes, the PRC1 and the PRC2 PcG complexes, which are essential for the maintenance of cell identity and play a role in oncogenesis. PcG complexes were recently identified as novel regulators of hematopoiesis, and appear to be expressed in a non‐overlapping pattern in resting and mature follicular B cells. Using highly specific antisera in combination with immunohistochemistry and triple immunofluorescence, we investigated the expression pattern of nine human PcG genes in germinal center (GC) B cells and highly purified germinal center B cell subpopulations. PcG proteins were detected in characteristic binding patterns that were not necessarily related to mutually exclusive expression of the two PcG complexes. We conclude that the two PcG complexes are expressed throughout GC development, and that the fine composition of each complex is determined by the differentiation status of the cell. In addition, a subset of dividing cells with a centrocyte CD marker profile was identified that co‐expresses core components of the PRC1 and PRC2 complex. We propose that these cells reflect a transitional stage between resting and dividing follicular B lymphocytes, and that they possibly represent the healthy precursors of nodal large B cell lymphomas.

Apoptosis in B-cell lymphomas and reactive lymphoid tissues always involves activation of caspase 3 as determined by a new in situ detection method.

In vitro studies indicate that in lymphomas, execution of apoptosis involves activation of effector caspases. To investigate activation of effector caspases in vivo in biopsy specimens of lymphomas, a new assay was developed using antibodies against active caspase 3 and p89, a protein fragment generated by caspase‐specific cleavage of poly‐ADP ribose polymerase (PARP). Using this assay, it was found that in B‐cell lymphomas, levels of active caspase 3/p89‐positive cells correlate strongly with morphologically recognizable apoptotic cells. The number of active caspase 3/p89‐positive cells was low in follicular lymphomas and usually high in diffuse large cell lymphomas. Highest numbers were found in Burkitt lymphomas and in two biopsies of diffuse large B‐cell lymphomas (DLCLs) obtained several days after initiation of therapy. It is concluded that apoptosis in reactive lymphoid tissues and in B‐cell lymphomas always involves activation of effector caspase 3 and cleavage of one of the major effector caspase substrates, PARP‐1. Moreover, levels of effector caspase activation are constantly low in low‐grade follicular lymphomas and vary considerably in DLCL and Burkitt lymphoma. Copyright

Percentage of activated cytotoxic T-lymphocytes in anaplastic large cell lymphoma and Hodgkin's disease: an independent biological prognostic marker

Recently, we demonstrated that the presence of high percentages of activated cytotoxic T-lymphocytes (CTLs) in biopsy specimens of both Hodgkins disease (HD) and ALK negative anaplastic large cell lymphoma (ALCL) is associated with a poor prognosis. To test whether this biological prognostic factor is more important in predicting clinical outcome than histological diagnosis or clinical factors, we compared the prognostic value of these parameters in an expanded group of classical HD and ALK negative ALCL. Tumor biopsies of classical HD (n = 83) and ALK negative systemic nodal ALCL (n = 43) were investigated for the presence of activated CTLs by immunohistochemistry, using a monoclonal antibody directed against granzyme B. Percentages of activated CTLs were quantified using Q-PRODIT, and their prognostic value was compared to that of histological diagnosis and clinical parameters, including age and stage. Both in classical HD and ALK negative ALCL, a high percentage of activated CTLs (ie ⩾15%) identified a group of patients with poor overall and progression-free survival time, even when adjusted for stage. In multivariate analysis, percentage of activated CTLs remained a strong independent prognostic marker, and was more sensitive than histological diagnosis or clinical factors in predicting overall survival time. We conclude that a high percentage of activated CTLs in the reactive infiltrate of ALK negative ALCL and classical HD is a strong indicator for an unfavorable clinical outcome, regardless of histological diagnosis or clinical parameters. As such, this biological parameter may be an especially helpful tool to determine therapeutic strategies in cases in which the differentiation between ALK negative ALCL and HD remains difficult.