IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool.

Immunotherapy with TCR-redirected T cells: comparison of TCR-transduced and TCR-engineered hematopoietic stem cell-derived T cells.

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However, transferred TCR chains can pair with endogenous TCR chains, resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity, because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice, TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study, we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner.

Protective capacity of virus-specific T cell receptor-transduced CD8 T cells in vivo.

The transfer of T cell receptor (TCR) genes by viral vectors represents a promising technique to generate antigen-specific T cells for adoptive immunotherapy. TCR-transduced T cells specific for infectious pathogens have been described, but their protective function in vivo has not yet been examined. Here, we demonstrate that CD8 T cells transduced with the P14 TCR specific for the gp33 epitope of lymphocytic choriomeningitis virus exhibit protective activities in both viral and bacterial infection models in mice.

TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo.

The hyaluronan-mediated motility receptor (HMMR/Rhamm) is overexpressed in numerous tumor types, including acute lymphoid leukemia and acute myeloid leukemia (AML). Several studies have reported the existence of T-cell responses directed against HMMR in AML patients that are linked to better clinical outcome. Therefore, we explored the use of HMMR-specific TCRs for transgenic expression in lymphocytes and their in vivo impact on HMMR(+) solid tumors and disseminated leukemia. We obtained TCRs via an in vitro priming approach in combination with CD137-mediated enrichment. Recipient lymphocytes expressing transgenic TCR revealed the specific tumor recognition pattern seen with the original T cells. Adoptive transfer experiments using a humanized xenograft mouse model resulted in significantly retarded solid tumor outgrowth, which was enhanced using IL-15-conditioned, TCR-transgenic effector memory cells. These cells also showed an increased potency to retard the outgrowth of disseminated AML, and this was further improved using CD8-enriched effector memory cells. To define a safe clinical setting for HMMR-TCR gene therapy, we analyzed transgenic T-cell recognition of hematopoietic stem cells (HSCs) and found on-target killing of HLA-A2(+) HSCs. Our findings clearly limit the use of HMMR-TCR therapy to MHC- mismatched HSC transplantation, in which HLA-A2 differences can be used to restrict recognition to patient HSCs and leukemia.

Targeting the epidermal growth factor receptor (HER) family by T cell receptor gene-modified T lymphocytes.

Human epidermal growth factor receptor 2 (HER2) has been successfully targeted as a breast cancer-associated antigen by various strategies. HER2 is also overexpressed in other solid tumors such as stomach cancer, as well as in hematological malignancies such as acute lymphoblastic leukemia. HER2-targeted therapies are currently under clinical investigation for a panel of malignancies. In this study, we isolated the T cell receptor (TCR) genes of a HER2-reactive allo-human leukocyte antigen-A2-restricted CTL clone and introduced the TCRα- and ß-chain genes into the retrovirus vector MP71. Murinization and codon optimization of the HER2-reactive TCR was required for efficient TCR expression in primary human T cells. The tumor recognition efficiency of HER2-TCR gene-modified T cells was similar to the parental CTL clone from which the TCR genes were isolated. The known cross-reactivity of the HER2-reactive TCR with HER3 and HER4 was retained when the TCR was transduced into primary T cells. Our results could contribute to the development of a TCR-based approach for the treatment of HER2-positive breast cancer, as well as of other malignancies expressing HER2, HER3, and/or HER4.

Necrotic death but not irradiation abolishes costimulation of T-cell effector functions and survival by CD80-expressing tumor cells.

Tumor vaccination by the use of gene-modified cancer cells that provide costimulatory signals has been successfully applied in preclinical animal models and is currently evaluated in a variety of clinical settings. In previous work, we demonstrated the efficacy of B7.1/CD80 to promote tumor immunity in syngeneic murine models and to prevent deletion of activated T cells by activation-induced cell death (AICD). In clinical trials, tumor cell vaccines are generally inactivated to avoid transfer of live tumor cells, i.e., additional tumor burden. Previous data indicated, however, that inactivation of tumor cells by lethal ionizing irradiation abrogates tumor vaccination by CD80-expressing cells. Here, we compare living and irradiated allogeneic tumor cells regarding their capacity to induce T-cell effector functions and their propensity to interfere with T-cell deletion by apoptosis. Both lethally irradiated and nonirradiated tumor cells facilitated T-cell proliferation, tumor cell lysis, and interfered with T-cell AICD to a similar extent. In contrast, necrotic tumor cells failed to costimulate T-cell effector functions. Thus, irradiation does not seem to hamper tumor cell-mediated costimulation of T-cell effector functions. In contrast, necrosis of gene-modified tumor cells abrogates costimulation of T cells by CD80-expressing cells.

Costimulation by CD137/4-1BB inhibits T cell apoptosis and induces Bcl-xL and c-FLIP(short) via phosphatidylinositol 3-kinase and AKT/protein kinase B.

Costimulation is essential for induction of T lymphocyte proliferation and inhibition of activation-induced cell death. While signaling pathways activated following the ligation of the costimulatory molecule CD28 are well defined, less is known about the molecular events induced by alternative costimulators. CD137/4-1BB, a costimulatory member of the tumor necrosis factor receptor family, plays an important role during late primary T cell stimulation. Here, we demonstrate for the first time that inhibition of activation-induced cell death by exposure to the CD137/4-1BB ligand involves up-regulation of the anti-apoptotic protein c-FLIP(short). Inhibition of T cell death by 4-1BB ligation and up-regulation of c-FLIP(short) and Bcl-x(L) were abolished by blocking the phosphatidylinositol 3-kinase or the AKT/protein kinase B, which also mediate CD28-induced inhibition of activation-induced cell death. Our findings, therefore, demonstrate that costimulatory molecules, although belonging to different protein families and participating in distinct upstream signaling pathways, employ common downstream signaling pathways.

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway.

Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.

Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway.

Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk.

Adenovirus-mediated overexpression of p14(ARF) induces p53 and Bax-independent apoptosis.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.

Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells.

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.

Inhibition of release of lentivirus particles with incorporated human influenza virus haemagglutinin by binding to sialic acid-containing cellular receptors.

Mutants of the haemagglutinin (HA) gene of human influenza virus A/Aichi/2/68 (H3N2) encoding HA proteins that are proteolytically cleaved intracellularly, defective in binding to cellular receptors or defective for acylation within the cytoplasmic C terminus have been generated. Here, the properties of these mutated HA molecules are described and their incorporation into the lipid membrane of released human immunodeficiency virus (HIV)-like particles is analysed. It is demonstrated that, when produced from cells coexpressing any of the binding-competent Aichi-HA molecules, release of HIV-like particles into the extracellular medium is reduced and the particles that are released fail to incorporate Aichi-HA. These blocks in release and incorporation, respectively, can both be overcome. The release of normal amounts of particles with incorporated HA can be achieved either by mutation of the receptor-binding site on the Aichi-HA molecule or by removal of sialic acid from surface proteins with neuraminidase. In contrast, as a result of blockage of the sialic acid-binding site by sialidated oligosaccharides on the HA itself, the HA of influenza virus A/FPV/Rostock/34 (H7N1) is efficiently incorporated into HIV-like particles. These results, namely that particle release can be inhibited by interactions between the incorporated glycoprotein and the cell surface and/or that interactions with other cellular components can be inhibitory to incorporation into retrovirus envelopes, probably reflect general principles that may hold for many viral and cellular glycoproteins.