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ABSTRACT: Persistent STAT3 signaling contributes to malignant progression in many diverse types of human cancer. STAT3 is constitutively active in activated B-cell (ABC)-like diffuse large B-cell lymphomas (DLBCL), a class of nongerminal center derived DLBCL cells for which existing therapy is weakly effective. In this report, we provide a preclinical proof of concept that STAT3 is an effective molecular target for ABC-like DLBCL therapy. Direct inhibition of STAT3 with short hairpin RNA suppressed the growth of human ABC-like DLBCL in mouse models in a manner associated with apoptosis, repression of STAT3 target genes, and inhibition of a tumor-promoting microenvironment. Together, these results suggest that STAT3 is essential to maintain the pathophysiology of ABC-like DLBCL and therefore that STAT3 inhibition may offer a promising approach in its therapy.
Cancer Research 05/2011; 71(9):3182-8. · 7.86 Impact Factor
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ABSTRACT: Although target cell cytolysis has been widely employed to describe effector function of cells, cytolysis assays as commonly employed do not generate quantitative data. In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and accurate, and comparable in sensitivity with the standard (51)Cr assay. We report on the potential for this assay to generate quantitative data on the cytolytic activity of both CD8 T and NK cells; describe a relationship between the efficiency of effector cell degranulation and target cell cytolysis throughout a range of E:T ratios, and demonstrate the potential to multiplex with other platforms to obtain broader datasets for the effector phenotype of cells. Appropriate use of this assay will enhance the ability to derive quantitative and integrated correlative datasets from basic, translational, and clinical studies.
Cytometry Part A 03/2010; 77(6):534-45. · 3.73 Impact Factor
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ABSTRACT: Two HLA-A*02-restricted epitopes have been identified within the VP1 polypeptide of a human polyomavirus, BK virus, which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Immunization of transgenic mice with recombinant modified vaccinia Ankara expressing BKV VP1 (rMVA-BKV VP1) elicited functional CTL populations recognizing the sequences LLMWEAVTV (amino acids residues 108-116, BKV VP1p108) and AITEVECFL (residues 44-52, BKV VP1p44) and cross-reactive to the previously described JC virus VP1 homologs. Flow-based analyses of PBMC from a panel of thirty healthy HLA-A*02 human volunteers indicated that the majority of these subjects harbored functional CTL populations recognizing the BKV epitopes and cross-reactive with the JCV homologs. CTL recognizing the JCV VP1p100 and JCV VP1p36 epitopes have previously been associated with prolonged survival in progressive multifocal leukoencephalopathy patients. These findings suggest that infection with BKV or JCV could potentially induce cross-protective T-cell immunity against diseases associated with these viruses.
Virology 07/2006; 350(1):128-36. · 3.35 Impact Factor
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ABSTRACT: A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127:1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168:499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71:115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.
Journal of Virology 10/2005; 79(17):11170-8. · 5.40 Impact Factor
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ABSTRACT: TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.
The Journal of Immunology 03/2005; 174(3):1188-95. · 5.79 Impact Factor
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ABSTRACT: Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.
European Journal of Immunology 11/2003; 33(10):2696-705. · 5.10 Impact Factor
