CD4+ T cells modulate the magnitude and durability of CTL responses in vivo and may serve as potent effector cells within the tumor microenvironment. The current study was undertaken to define novel
...epitopes from the broadly expressed tumor antigen MAGE-6 that are recognized by CD4+ T cells. We have combined the use of
a HLA-DR4/peptide binding algorithm with the IFN-γ enzyme-linked immunospot assay to identify four nonoverlapping sequences
derived from the MAGE-6 protein that served as CD4+ T-cell epitopes in HLA-DR4+ donors. Strikingly, patients with active melanoma
or renal cell carcinoma failed to secrete IFN-γ in response to MAGE-6-derived epitopes, whereas both normal donors and cancer
patients with no current evidence of disease were responsive, particularly after short-term in vitro stimulations with peptide-pulsed dendritic cells. Importantly, peptide-specific CD4+ T cells also recognized HLA-DRβ1*0401+
tumor cells that constitutively expressed the MAGE-6 protein and autologous HLA-DRβ1*0401+ dendritic cells transfected with
MAGE-6 cDNA-elicited CD4+ T cells that reacted against individual peptide epitopes in vitro . These data suggest that MAGE-6-derived epitopes could serve as useful vaccine candidate components and may provide an immune-monitoring
index of clinically important Th1-type immunity in patients with renal cell carcinoma or melanoma.
It is now understood that the genetic plasticity of cancer cells can lead to alterations that confer selective growth advantages to the tumor, some of which play a role in immune escape. A number of ...mutations veiling tumor cells from host immune defenses have been well characterized but more recent studies suggest that a variety of tumors can also express products that are actually toxic for the immune effectors. A component of this tumor-induced T-cell death has been attributed to receptor-mediated apoptosis. Some tumors, however, synthesize soluble factors that mediate similar effects. In this regard, we previously showed that supernatants from explanted renal cell carcinoma (RCC) tumors sensitized normal T cells to activation induced cell death, and the responsible products had the features of gangliosides. We have also shown that renal tumor lines, including SK-RC-45, induce apoptosis of both Jurkat cells and normal T lymphocytes. Here, we used the ganglioside synthesis inhibitor PPPP to define the role of gangliosides in RCC cell line (SK-RC-45)-mediated T cell and Jurkat cell apoptosis and to elucidate the proapoptotic molecular events by which the glycosphingolipids produce their effects. The ganglioside-synthesizing SK-RC-45 line stimulated the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) positivity of cocultured T cells by a mechanism that involved decreasing lymphocyte expression levels of Bcl-2 and Bcl-(XL), inducing cytochrome c release from their mitochondria and activating caspases 9 and 3. These proapoptotic events were partially or completely abrogated when tumor cells were pretreated with PPPP for 5 days before the SK-RC-45/T lymphocyte coincubation, a regimen that reduced tumor-associated ganglioside levels by 70-80%. Our results suggest that gangliosides may be key mediators of RCC-induced T-cell apoptosis and imply that they contribute to the T-cell dysfunction in the tumor microenvironment.
Tumors may escape immune recognition and destruction through the induction of apoptosis in activated T lymphocytes. Results
from several laboratories suggest that FasL (L/CD95L) expression in tumors ...may be responsible for this process. In this study
of patients with renal cell carcinoma (RCC), we provide evidence for two mechanisms of T-cell apoptosis. One mechanism involves
the induction of apoptosis via FasL expression in tumor cells. This is supported by several observations, including the fact
that tumor cells in situ as well as cultured cell lines expressed FasL mRNA and protein by a variety of techniques. The FasL in RCC is functional
because in coculture experiments, FasL + tumors induced apoptosis in Fas-sensitive Jurkat T cells and in activated peripheral blood T cells but not in resting peripheral
blood T cells. Most importantly, antibody to FasL partially blocked apoptosis of the activated T cells. Moreover, Fas was
expressed by T cells derived from the peripheral blood (53% median) and tumor (44.3% median) of RCC patients. Finally, in situ staining for DNA breaks demonstrated apoptosis in a subset of T cells infiltrating renal tumors. These studies also identified
a second mechanism of apoptosis in RCC patient peripheral T cells. Whereas these cells did not display DNA breaks when freshly
isolated or after culture for 24 h in medium, peripheral blood T cells from RCC patients underwent activation-induced cell
death after stimulation with either phorbol 12-myristate 13-acetate/ionomycin or anti-CD3/CD28 antibodies. Apoptosis mediated
by exposure to FasL in tumor cells or through T-cell activation may contribute to the failure of RCC patients to develop an
effective T-cell-mediated antitumor response.
T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which ...in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.
Antitumor immunity fails to adequately develop in many cancer patients, including those with renal cell carcinoma (RCC).
A number of different mechanisms have been proposed to explain the immune ...dysfunction observed in cancer patient T cells.
Here we show that T cells from RCC patients display increased sensitivity to apoptosis. Tumor-infiltrating lymphocytes (TILs)
display the most profound sensitivity, because 10–15% of those cells are apoptotic when assessed by terminal deoxynucleotidyltransferase-mediated
nick end labeling in situ , and the number of apoptotic TILs further increases after 24 h of culture. Peripheral blood T cells from RCC patients are
not directly apoptotic, although T lymphocytes derived from 40% of those individuals undergo activation-induced cell death
(AICD) upon in vitro stimulation with phorbol myristate acetate and ionomycin. This is in contrast to T cells from normal individuals, which are
resistant to AICD. TILs and peripheral blood T cells from RCC patients also exhibit impaired activation of the transcription
factor, nuclear factor (NF)-κB. Additional findings presented here indicate that the heightened sensitivity of patient T cells
to apoptosis may be tumor induced, because supernatants from RCC explants sensitize, and in some instances directly induce,
normal T cells to apoptosis. These same supernatants also inhibit NF-κB activation. RCC-derived gangliosides may represent
one soluble tumor product capable of sensitizing T cells to apoptosis. Pretreatment with neuraminidase, but not proteinase
K, abrogated the suppressive effects of tumor supernatants on both NF-κB activation and apoptosis. Additionally, gangliosides
isolated from tumor supernatants not only inhibited NF-κB activation but also sensitized T cells to AICD. These findings demonstrate
that tumor-derived soluble products, including gangliosides, may contribute to the immune dysfunction of T cells by altering
their sensitivity to apoptosis.
Apoptotic DNA fragmentation minimizes the risk of transferring genetic information from apoptotic cancer cells to the neighboring cells. We have reported previously that caspase-deficient human renal ...cell carcinoma (RCC) lines were almost completely resistant to apoptosis in response to cytotoxic agents. In the present report we examined apoptotic process in caspase competent RCC-91 cells. Apoptosis in RCC-91 cells was accompanied by activation of caspases-3 and -9; cleavage of PARP and DFF45 proteins; typical apoptotic nuclei fragmentation and mitochondrial collapse. Nevertheless, DNA in these cells was not degraded into oligonucleosomal fragments compared to control Jurkat cells. Expression of caspase-activated DNase, DFF40 accountable for characteristic ladder pattern was easily detectable in Jurkat but not renal cancer cells, providing one possible explanation for the lack of oligonucleosomal DNA fragmentation in apoptotic RCC cells. Lack of typical DNA fragmentation indicates a potential threat of transferring genetic information from one tumor cell to another or to the neighboring healthy cells.
We report on the role of hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared with ...matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared with primary mammary tumors in a mouse model of metastatic breast cancer, the polyoma middle T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF(-)1α protein allows not only for inhibition of vascular endothelial growth factor-regulated angiogenesis, but also for inhibition of compensatory pro-angiogenic pathways and recruitment of bone marrow-derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion that can be partly attributed to decreased membrane localization of the 67 kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus, HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.
A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs ...containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.
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