Biomedical subjects

M J Smyth

Publications and source records attributed to M J Smyth.

At least 19 recordsLinked to original sources

PubMed2000

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL.

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.

Adenocarcinoma↗

PubMed2000

HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance.

Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein. As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation. However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B. It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P-gp-mediated MDR. We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and -negative cell lines at concentrations of 10 mmol/L and above. The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels. In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected. These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Differential tumor surveillance by natural killer (NK) and NKT cells.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

Animals↗

PubMed2000

Tumor necrosis factor sustains the generalized lymphoproliferative disorder (gld) phenotype.

Tumor necrosis factor (TNF) and Fas ligand (FasL) play major roles in the homeostasis of the peripheral immune system. This becomes dramatically obvious in the absence of a functional FasL. Mice with such a deficiency develop a profound lymphadenopathy, splenomegaly, hypergammaglobulinemia, and strain-dependent systemic autoimmune disease, and succumb to premature death. It is consequently termed generalized lymphoproliferative disorder (gld). By contrast, TNF deficiency alone does not result in a striking phenotype. Thus, we sought to determine what role TNF might play in contributing to the gld phenotype by creating C57BL/6.gld.TNF(-/-) mice. Contrary to the expected outcome, mice deficient for both FasL and TNF had a substantially milder gld phenotype with regard to mortality, lymphoaccumulation, germinal center formation, and hypergammaglobulinemia. To confirm these data in a strain highly permissive for the phenotype, C3H/HeJ.gld and C3H.HeJ.lpr mice were treated with a TNF-specific monoclonal antibody. This transient neutralization of TNF also resulted in a significantly attenuated lymphoproliferative phenotype. We conclude that TNF is necessary for the full manifestation of the lymphoproliferative disorder, in particular playing a critical role in lymphoaccumulation. Most importantly, absence of TNF protects gld mice against premature death.

Animals↗

PubMed2000

Multiple physiological functions for multidrug transporter P-glycoprotein?

Multidrug resistance mediated by the drug-efflux protein P-glycoprotein (P-gp) is one mechanism that tumor cells use to escape death induced by chemotherapeutic drugs. Although it is irrefutable that P-gp can efflux xenobiotics out of cells, biological regulatory functions for P-gp in multicellular organisms have yet to be established firmly. Recent observations have challenged the notion that P-gp has evolved merely to efflux xenotoxins out of healthy cells and raised the possibility that P-gp and related transporter molecules might play a fundamental role in regulating cell differentiation, proliferation and survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Aberrant sphingolipid signaling is involved in the resistance of prostate cancer cell lines to chemotherapy.

Activation of the apoptosis program has been implicated in the response of cancer cells to chemotherapy. Therefore, we postulated that chemotherapy-resistant prostate cancer has developed a lesion in the apoptosis signal transduction cascade. In this study, we investigated the mechanism underlying the resistance of apoptosis-insensitive prostate cancer cells to apoptosis. We approached this by comparing the response of the androgen-sensitive LNCaP cell line and the androgen-insensitive PC3 cell line to treatment with the topoisomerase I inhibitor, camptothecin. We demonstrated that LNCaP cells are susceptible to camptothecin-induced cell death, and PC3 cells are resistant. Additional studies confirmed that the mode of cell death in the LNCaP cells was by apoptosis. We then determined that a component of the resistance to death in the apoptosis-insensitive cells involved a defect in the generation of ceramide, a key lipid mediator of apoptosis. Specifically, we demonstrated that PC3 cells are unable to elevate ceramide in response to treatment with camptothecin. In contrast, elevations in ceramide levels occur in LNCaP cells in response to the same treatment. Significantly, additional studies showed that treatment with exogenous ceramide overcomes the lesion in the PC3 cells and induces apoptosis. In attempting to gain preliminary insight into the nature of the lesion in ceramide formation in the apoptosis-resistant cells, we established that generation of ceramide in LNCaP cells is independent of the de novo pathway. These studies present novel insights into the mechanism by which prostate cancer cells may be resistant to induction of apoptosis. The significance of this study lies in the fact that an understanding of the biological and molecular events contributing to the resistance of prostate cancer to therapy is crucial to the development of more effective regimens for advanced disease.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Interleukin 2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat)5 activation of two enhancers.

Optimal T cell differentiation into effector cells with specialized functions requires the participation of cytokine receptor signals. In T helper cells, this process is controlled by chromatin changes and distal and proximal regulatory elements as well as specific transcription factors. Analogous events during cytotoxic T lymphocyte (CTL) differentiation remain to be identified. This process is known, however, to be crucially regulated by interleukin (IL)-2 receptor (R) signals. It is accompanied by the induction of perforin expression via a mechanism that does not entail proximal regulatory elements. In this report, transgenically expressed human perforin gene locus DNAs demonstrate that IL-2R signals target two IL-2-dependent enhancers approximately 15 and 1 kilobase upstream of the promoter. The most distal enhancer may also respond to TCR signals. In transient transfections, both enhancers required two identically spaced Stat-like elements for their activation, which was abolished by expression of a dominant negative signal transducer and activator of transcription (Stat)5 molecule, whereas a constitutively active Stat5 molecule bypassed the requirement for IL-2R signals. These results provide a molecular explanation for the activation of the perforin gene during CTL differentiation and complement the analysis of animals deficient in the activation of the IL-2R Stat signaling pathway by establishing perforin as a target gene.

Animals↗

PubMed1999

Accessory function for NK1.1+ natural killer cells producing interferon-gamma in xenospecific cytotoxic T lymphocyte differentiation.

BACKGROUND: We have previously demonstrated that xenospecific cytotoxic T lymphocyte (CTL) differentiation requires accessory function by NK1.1+ cells, yet the mechanism by which NK1.1+ cells support CTL generation had not been elucidated. METHODS: An established model in which mice generate a strong local popliteal lymph node CTL response to footpad immunizations with human tumor cells was used. Mice depleted of NK1.1+ cells fail to mount a maximal xenospecific CD8+ CTL response. The xenospecific CTL response in anti-NK1.1 monoclonal antibody-depleted mice could be completely restored if mice were coinoculated with human tumor cells (the xenoantigen) and xenoantigen-stimulated syngeneic natural killer (NK) cells from wild-type or perforin-deficient mice. By contrast, NK1.1+ cells from interferon-gamma-deficient mice did not restore the maturation of xenospecific CTL in anti-NK1.1 monoclonal antibody-treated mice. Depletion of NK1.1+ cells in vivo from both wild-type and Jalpha281-deficient mice (which lack Valpha14 NK1.1+ T cells) abrogated the generation of xenospecific CTL, however, untreated Jalpha281-deficient mice mounted a normal xenogeneic response. CONCLUSIONS: These data indicate that local NK cell production of interferon-gamma at the site of challenge is an important stimulus for generating xenospecific CTL in local draining lymph nodes and that Valpha14 NK T cells play little or no regulatory function in this response.

Animals↗

PubMed1999

Perforin-dependent cytolytic responses in beta2-microglobulin-deficient mice.

The ability of beta2-microglobulin-deficient mice (B6.beta2micro(o)) mice to reject syngeneic and major histocompatability (MHC) class I-deficient tumor grafts was examined with a view to determining residual cytotoxic activities that exist in these mice. In particular, the cytotoxic activities of NK cells and CD8(+) cytotoxic T lymphocytes (CTL) reactive against self-MHC class I were assessed using a variety of gene-targeted mice. The creation of mice doubly deficient for perforin and beta2micro (B6.P(o).beta2micro(o)) enabled the determination that perforin was responsible for the cytotoxic activity of NK cells and CD8(+) CTL reactive against self-MHC class I. Dependence on perforin function was demonstrated for the cytotoxicity of these effectors in vitro and for the ability of these effectors to reject a variety of tumors in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection.

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Animals↗

PubMed1999

Multiple deficiencies underlie NK cell inactivity in lymphotoxin-alpha gene-targeted mice.

We have evaluated the NK cell antitumor activity in lymphotoxin (LT)-deficient mice. Both NK cell-mediated tumor rejection and protection from experimental metastases were significantly compromised in LT-alpha-deficient mice. Analysis of LT-alpha-deficient mice revealed that the absolute number of alphabetaTCR- NK1.1+ NK cells was reduced in bone marrow and thymus, but with overall proportional decreases in other hemopoietic organs. In addition, the antitumor potential of alphabetaTCR- NK1.1+ cells, as determined by their lytic capacity and perforin expression, was reduced 1.5- to 3-fold in LT-alpha-deficient mice, as compared with wild-type mice. Combined defects in NK cell development and effector function contribute to compromised NK cell antitumor function in LT-alpha-deficient mice.

Animals↗

PubMed1999

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.

Animals↗

PubMed1999

P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death.

A major problem with treating patients with cancer by traditional chemotherapeutic regimes is that their tumors often develop a multidrug resistant (MDR) phenotype and subsequently become insensitive to a range of different chemotoxic drugs. One cause of MDR is overexpression of the drug-effluxing protein, P-glycoprotein. It is now apparent that P-glycoprotein may also possess a more generic antiapoptotic function that protects P-glycoprotein-expressing cancer cells and normal cells from cell death. Herein we show that cells induced to express P-glycoprotein either by drug selection or by retroviral gene transduction with MDR1 cDNA are resistant to cell death induced by a wide range of death stimuli, such as FasL, tumor necrosis factor (TNF), and ultraviolet (UV) irradiation, that activate the caspase apoptotic cascade.However, P-glycoprotein-expressing cells were not resistant to caspase-independent cell death mediated by pore-forming proteins and granzyme B.MDR P-glycoprotein-expressing cells were made sensitive to caspase-dependent apoptosis by the addition of anti-P-glycoprotein antibodies or verapamil, a pharmacological inhibitor of P-glycoprotein function. Clonogenic assays showed that P-glycoprotein confers long-term resistance to caspase-dependent apoptotic stimuli but not to caspase-independent cell death stimuli. This study has confirmed a potential novel physiological function for P-glycoprotein and it now remains to dissect the molecular mechanisms involved in the inhibition of capsase-dependent cell death by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

NKT cells are phenotypically and functionally diverse.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

Animals↗

PubMed1999

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes.

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor gamma chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-gamma clones, and lysis of erbB-2+ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-gamma, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL.

Animals↗

PubMed1999

CTL granules: evolution of vesicles essential for combating virus infections.

Viral strategies for escaping apoptosis have co-evolved with the immune system, resulting in a complex balance of pro- and anti-apoptotic forces in virus-infected cells under attack by cytotoxic T lymphocytes (CTLs). Here, Joseph Trapani and colleagues argue that CTL cytolytic granules are the principal apoptotic means of eliminating viruses and possess multiple independent mechanisms to counter the viral anti-apoptotic machinery.

Animals↗

PubMed1999

NK cells and apoptosis.

Natural killer (NK) cells are a cell of the innate immune system that play an important role in the early response to viral infections and tumours. Natural killer cells are cytolytic, and secrete cytokines that influence the developing antigen-specific immune response. In the present article the NK cell surface molecules regulating effector function, the NK cell effector mechanisms involved in apoptosis, and the role of NK cell effector mechanisms in immune responses are reviewed.

Animals↗

PubMed1998

An essential role for tumor necrosis factor in natural killer cell-mediated tumor rejection in the peritoneum.

Natural killer (NK) cells are thought to provide the first line of defence against tumors, particularly major histocompatibility complex (MHC) class I- variants. We have confirmed in C57BL/6 (B6) mice lacking perforin that peritoneal growth of MHC class I- RMA-S tumor cells in unprimed mice is controlled by perforin-dependent cytotoxicity mediated by CD3(-) NK1.1(+) cells. Furthermore, we demonstrate that B6 mice lacking tumor necrosis factor (TNF) are also significantly defective in their rejection of RMA-S, despite the fact that RMA-S is insensitive to TNF in vitro and that spleen NK cells from B6 and TNF-deficient mice are equally lytic towards RMA-S. NK cell recruitment into the peritoneum was abrogated in TNF-deficient mice challenged with RMA-S or RM-1, a B6 MHC class I- prostate carcinoma, compared with B6 or perforin-deficient mice. The reduced NK cell migration to the peritoneum of TNF-deficient mice correlated with the defective NK cell response to tumor in these mice. By contrast, a lack of TNF did not affect peptide-specific cytotoxic T lymphocyte-mediated rejection of tumor from the peritoneum of preimmunized mice. Overall, these data show that NK cells delivering perforin are the major effectors of class I- tumor rejection in the peritoneum, and that TNF is specifically critical for their recruitment to the peritoneum.

Animals↗