Brian R. Gastman

Lymphocyte apoptosis induced by Fas ligand- expressing ovarian carcinoma cells. Implications for altered expression of T cell receptor in tumor-associated lymphocytes.

We have recently reported that tumor-associated lymphocytes obtained from ascitic fluids of women with ovarian carcinoma (OvCA) demonstrate a marked decrease in expression of cytoplasmic CD3-zeta and surface CD3-epsilon chains, which is associated with altered function of T cell receptor (TcR). We now demonstrate that OvCAs in situ and in culture express functional Fas ligand (FasL), capable of triggering an intrinsic cell death program in Fas-expressing T cells. The possibility of a relationship between cell death and altered expression of TcR was examined. The data indicate that alterations in expression of CD3-zeta and CD3-epsilon chains in T cells coincubated with OvCA are related to tumor-induced apoptosis, as the addition of pan-caspase inhibitors, DEVD-cho or YVAD-cho, prevents both the in vitro induction of T cell death by OvCA cells and the changes in the level of expression of CD3-zeta and CD3-epsilon chains. In the presence of Fas-Fc fusion protein, but not Fc-control protein, the loss in expression of CD3-zeta and CD3-epsilon chains induced in T cells by FasL+ OvCA cells was prevented. These results suggest that the loss in expression of CD3-zeta and CD3-epsilon chains in T lymphocytes interacting with OvCA cells is associated with apoptosis mediated by FasL-expressing tumor cells.

Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis.

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Tumor's other immune targets: dendritic cells.

The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co‐culture of murine or human DC with different tumor cell lines for 4–48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase‐3‐like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time‐ and dose‐dependent induction of apoptosis in DC by tumor‐derived factors. This finding is both effector and target specific. The mechanism of tumor‐induced DC apoptosis involved regulation of Bcl‐2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor‐associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC‐based immunotherapies for cancer and other immune diseases. J. Leukoc. Biol. 66: 336–344; 1999.

Human γδ T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement

Natural killer (NK) cells are innate effector lymphocytes that control the growth of major histocompatibility complex class I negative tumors. We show here that γδ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell-mediated killing of tumors that are usually resistant to NK cytolysis. The induction of cytotoxicity toward these resistant tumors requires priming of NK cells by immobilized human immunoglobulin G1 and costimulation through CD137L expressed on activated γδ T lymphocytes. This costimulation increases NKG2D expression on the NK-cell surface, which is directly responsible for tumor cell lysis. Moreover, culturing peripheral blood mononuclear cells with zoledronic acid, a γδ T lymphocyte activating agent, enhances NK-cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors. Our data reveal a novel function of human γδ T lymphocytes in the regulation of NK cell-mediated cytotoxicity and provide rationale for the use of strategies to manipulate the CD137 pathway to augment innate antitumor immunity.

Isopentenyl Pyrophosphate–Activated CD56+ γδ T Lymphocytes Display Potent Antitumor Activity toward Human Squamous Cell Carcinoma

Purpose: The expression of CD56, a natural killer cell–associated molecule, on αβ T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of γδ T cells. However, antitumor effector functions of CD56+ γδ T cells are poorly characterized. Experimental Design: To investigate the potential effector role of CD56+ γδ T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2–expanded γδ T cells from peripheral blood mononuclear cells of healthy donors. Results: Thirty to 70% of expanded γδ T cells express CD56 on their surface. Interestingly, although both CD56+ and CD56− γδ T cells express comparable levels of receptors involved in the regulation of γδ T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56+ γδ T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56+ γδ T lymphocytes expressed higher levels of CD107a compared with CD56− controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-γδ T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56+ γδ T cells involves the perforin-granzyme pathway and is mainly γδ T-cell receptor/NKG2D dependent. Importantly, CD56-expressing γδ T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Conclusions: Our data indicate that CD56+ γδ T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

Degradation of Mcl-1 by Granzyme B IMPLICATIONS FOR Bim-MEDIATED MITOCHONDRIAL APOPTOTIC EVENTS

Recent studies have suggested that in the absence of Bid, granzyme B (GrB) can utilize an unknown alternative pathway to mediate mitochondrial apoptotic events. The current study has elucidated just such a pathway for GrB-mediated mitochondrial apoptotic alterations. Two Bcl-2 family members have been identified as interactive players in this newly discovered mitochondrial response to GrB: the pro-survival protein Mcl-1L and the pro-apoptotic protein, Bim. Expression of Mcl-1L, which localizes mainly to the outer mitochondrial membrane, decreases significantly in cells subjected to CTL-free cytotoxicity mediated by a combination of GrB and replication-deficient adenovirus. The data suggest that Mcl-1L is a substrate for GrB and for caspase-3, but the two enzymes appear to target different cleavage sites. The cleavage pattern of endogenous Mcl-1L resembles that of in vitro translated Mcl-1L subjected to similar proteolytic activity. Co-immunoprecipitation experiments performed with endogenous as well as with in vitro translated proteins suggest that Mcl-1L is a high affinity binding partner of the three isoforms of Bim (extra-long, long, and short). Bim, a BH3-only protein, is capable of mediating the release of mitochondrial cytochrome c, and this activity is inhibited by the presence of exogenous Mcl-1L. The findings presented herein imply that Mcl-1L degradation by either GrB or caspase-3 interferes with Bim sequestration by Mcl-1L.

What is the best surgical margin for a Basal cell carcinoma: a meta-analysis of the literature.

Background: Current management of basal cell carcinoma is surgical excision. Most resections use predetermined surgical margins. The basis of ideal resection margins is almost completely from retrospective data and mainly from small case series. This article presents a systematic analysis from a large pool of data to provide a better basis of determining ideal surgical margin. Methods: A systematic analysis was performed on data from 89 articles from a larger group of 973 articles selected from the PubMed database. Relevant inclusion and exclusion criteria were applied to all articles reviewed and the data were entered into a database for statistical analysis. Results: The total number of lesions analyzed was 16,066; size ranged from 3 to 30 mm (mean, 11.7 ± 5.9 mm). Surgical margins ranged from 1 to 10 mm (mean, 3.9 ± 1.4 mm). Negative surgical margins ranged 45 to 100 percent (mean, 86 ± 12 percent). Recurrence rates for 5-, 4-, 3-, and 2-mm surgical margins were 0.39, 1.62, 2.56, and 3.96 percent, respectively. Pooled data for incompletely excised margins have an average recurrence rate of 27 percent. Conclusions: A 3-mm surgical margin can be safely used for nonmorpheaform basal cell carcinoma to attain 95 percent cure rates for lesions 2 cm or smaller. A positive pathologic margin has an average recurrence rate of 27 percent.

Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis.

Recently, we reported the identification of a novel mitochondrial apoptotic pathway for granzyme B (GrB) (Han, J., Goldstein, L. A., Gastman, B. R., Froelich, C. J., Yin, X. M., and Rabinowich, H. (2004) J. Biol. Chem. 279, 22020–22029). The newly identified GrB-mediated mitochondrial cascade was initiated by the cleavage and subsequent degradation of Mcl-1, resulting in the release of mitochondrial Bim from Mcl-1 sequestration. To investigate the biological significance of Mcl-1 cleavage by GrB, we mapped the major GrB cleavage sites and evaluated the apoptotic potential of the cleavage products. GrB cleaves Mcl-1 after aspartic acid residues 117, 127, and 157, generating C-terminal fragments that all contain BH-1, BH-2, BH-3, and transmembrane domains. These fragments accumulate at an early apoptotic phase but are eliminated by further degradation during the apoptotic process. The major Mcl-1 C-terminal fragment generated by GrB (residues 118–350) was unable to induce or enhance apoptosis when transfected into tumor cells. Instead, this Mcl-1 C-terminal fragment maintained a partial protective capability against GrB-mediated apoptosis via its lower affinity to Bim. In comparison with ectopically expressed full-length Mcl-1, the stably transfected C-terminal fragments of Mcl-1 were less efficiently localized to the mitochondria. Knockdown of Mcl-1, as achieved by transfection with Mcl-1-specific short interfering RNA, resulted in a significant level of apoptosis in the absence of external apoptotic stimulation and, in addition, enhanced the susceptibility of breast carcinoma cells to GrB cytotoxicity. The significance of Bim in this GrB apoptotic cascade was indicated by the marked protection against GrB-mediated apoptosis endowed on these cells through Bim knockdown. Our studies suggest that the disruption of the Mcl-1·Bim complex by GrB initiates a major Bim-mediated cellular cytotoxic mechanism that requires the elimination of Mcl-1 following its initial cleavage.

Tumor-Induced Senescent T Cells with Suppressor Function: A Potential Form of Tumor Immune Evasion

Senescent and suppressor T cells are reported to be increased in select patients with cancer and are poor prognostic indicators. Based on the association of these T cells and poor outcomes, we hypothesized that tumors induce senescence in T cells, which negatively effects antitumor immunity. In this report, we show that human T cells from healthy donors incubated with tumor for only 6 h at a low tumor to T-cell ratio undergo a senescence-like phenotype, characterized by the loss of CD27 and CD28 expression and telomere shortening. Tumor-induced senescence of T cells is induced by soluble factors and triggers increases in expression of senescence-associated molecules such as p53, p21, and p16. Importantly, these T cells are not only phenotypically altered, but also functionally altered as they can suppress the proliferation of responder T cells. This suppression requires cell-to-cell contact and is mediated by senescent CD4(+) and CD8(+) subpopulations, which are distinct from classically described natural T regulatory cells. Our observations support the novel concept that tumor can induce senescent T cells with suppressor function and may effect both the diagnosis and treatment of cancer.

CD137 Promotes Proliferation and Survival of Human B Cells

CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig–stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-γ and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-α and -β. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.