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ABSTRACT: Cytotoxic lymphocytes and dendritic cells infiltrating human renal cell carcinoma (RCC) are not sufficient to prevent tumor progression. Our studies identified alterations of the immune cell infiltrate as well as some of the underlying mechanisms. This knowledge should facilitate the development of anti-RCC therapies that achieve better tumor control.
Oncoimmunology. 11/2012; 1(8):1451-1453.
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ABSTRACT: Tissue inhibitors of metalloproteinases exhibit diverse physiological/biological functions including moderation of the proteolytic processing of growth factors and turnover of extracellular matrix. These various biological activities are linked in part to the stoichiometry of tissue inhibitor of metalloprotein/matrix metalloprotein (TIMP/MMP)/surface protein interactions. TIMP-1, a secreted protein, can be detected on the cell surface only through its interaction with surface-bound proteins. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells or tissues, are efficiently incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to focus defined concentrations of the inhibitory protein independently on the surface of primary dermal fibroblast cells. Exogenously added recombinant TIMP-1-GPI effectively inserted into the cell membrane of fibroblasts blocked the secretion of MMPs and markedly altered the stoichiometry of MMP association with the cell surface. TIMP-1-GPI treatment resulted in inhibition of fibroblast-reduced proliferation, and transiently reduced expression of fibrosis-associated genes. These effects were dose dependent. Treated cells also showed a more proapoptotic phenotype based on apoptotic assays and western blot analysis for apoptosis-associated protein expression. GPI-anchored TIMP-1 may represent a more effective version of the protein for use in therapeutic approaches to help control fibrosis and scar formation.Journal of Investigative Dermatology advance online publication, 25 October 2012; doi:10.1038/jid.2012.375.
Journal of Investigative Dermatology 10/2012; · 6.31 Impact Factor
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ABSTRACT: CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.
The Journal of Immunology 05/2012; 188(12):5990-6000. · 5.79 Impact Factor
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ABSTRACT: Adding hyperthermia to chemotherapy improved the clinical outcome of patients with high risk soft tissue sarcoma. Further improvement might be possible if combined with vaccination strategies. As no sarcoma-associated antigens are known, the ectopic expression of a surrogate marker for which immune monitoring tools are available, is envisaged. We tested surrogate marker transfer into sarcoma cells in vitro using modified vaccinia virus Ankara (MVA), which has well established clinical safety. We examined its robustness against standard sarcoma treatment modalities, such as ifosfamide and hyperthermia.
We transduced sarcoma cell lines and primary tumour cells from sarcoma patients with MVA encoding the human tyrosinase gene (MVA-hTyr). Kinetics of tyrosinase expression and the potency to activate tyrosinase-specific cytotoxic T cells were assessed. In addition cells were exposed to chemotherapy and heat, imitating the clinical setting.
Tyrosinase was ectopically expressed in sarcoma cells. Infected cells presented tyrosinase epitopes for T cell recognition even if exposed to ifosfamide/heat.
As sarcoma patients receive surgery up front or after neoadjuvant systemic chemotherapy/hyperthermia, tumour material is generally available. Our data document that primary sarcoma cells can be infected with MVA-hTyr in vitro and antigen presentation is not affected by ifosfamide or heat treatment. Infected cells can serve as a source for vaccine preparation. MVA-hTyr infection of tumour cells lacking defined antigens is a feasible system to introduce a robust surrogate marker to provide an immune monitoring marker for assessing the induction of antigen-specific T cell activation.
International Journal of Hyperthermia 01/2012; 28(1):33-42. · 1.92 Impact Factor
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ABSTRACT: Lactic acidosis is common to most solid tumors and has been found to affect infiltrating immune cells. Here we document effector phase inhibition of cytotoxic T cells (CTLs) involving complete blockage of cytokine production and partial impairment of lytic granule exocytosis. Lactic acidosis impaired TCR-triggered phosphorylation of JNK, c-Jun and p38, while not affecting MEK1 and ERK. The select targeting of signaling proteins involved in IFNγ production (JNK/c-Jun, p38) without affecting those jointly used in cytokine regulation and granule exocytosis (MEK1/ERK) explains the observed split effect of lactic acidosis on the CTL responses. CTL inhibition by lactic acidosis showed fast dynamics with immediate onset and reversion. Functional recovery by neutralizing the extracellular pH despite continuous presence of lactate holds promise that CTL activity can be improved in the milieu of solid tumors with appropriate anti-acidosis treatment, thereby increasing the efficacy of adoptive T cell therapy.
International Journal of Cancer 09/2011; 131(3):633-40. · 5.44 Impact Factor
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ABSTRACT: Clear cell renal cell carcinoma (ccRCC) is an aggressive and difficult to manage cancer. Immunotherapy has the potential to induce long-lasting regression in a small group of patients. However, severe side effects limit broad application which highlights the need for a marker to distinguish responder from nonresponder. TNMG staging, referring to tumor size, lymph node involvement, presence of metastasis, and grade of tumor differentiation, represents an important prognostic system but is not useful for predicting responders to immunotherapy. NK cells are potent antitumor effector cells, and a role as prognostic marker in some solid tumors has been suggested. As NK cells are responsive to various immune modifiers, they may be important mediators of patient response to immunotherapies, in particular those including IL-2. We report that the NK cell percentage within RCC-infiltrating lymphocytes, as determined by flow cytometry, allows ccRCC subgrouping in NK(high)/NK(low) tissues independent of TNMG classification. Quantitative reverse transcriptase polymerase chain reaction using whole-tissue RNA identified four markers (NKp46, perforin, CX(3)CL1, and CX(3)CR1) whose transcript levels reproduced the NK(high)/NK(low) tissue distinction identified by flow cytometry with high selectivity and specificity. Combined in a multiplex profile and analyzed using neural network, the accuracy of predicting the NK(high)/NK(low) groups was 87.8%, surpassing that of each single marker. The tissue transcript signature, based on a robust high-throughput methodology, is easily amenable to archive material and clinical translation. This now allows the analysis of large patient cohorts to substantiate a role of NK cells in cancer progression or response to immunotherapy.
Journal of Molecular Medicine 08/2011; 90(1):55-66. · 4.67 Impact Factor
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Ainhoa-M Figel,
Dorothee Brech,
Petra U Prinz,
Ulrike K Lettenmeyer,
Judith Eckl,
Adriana Turqueti-Neves,
Josef Mysliwietz,
David Anz,
Nicole Rieth,
Niklas Muenchmeier,
Alexander Buchner,
Stefan Porubsky,
Sabine I Siegert,
Stephan Segerer,
Peter J Nelson, Elfriede Noessner
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ABSTRACT: Tissue dendritic cells (DCs) may influence the progression of renal cell carcinoma (RCC) by regulating the functional capacity of antitumor effector cells. DCs and their interaction with T cells were analyzed in human RCC and control kidney tissues. The frequency of CD209(+) DCs in RCCs was found to be associated with an unfavorable T(H)1 cell balance in the tissue and advanced tumor stages. The CD209(+) DCs in RCC were unusual because most of them co-expressed macrophage markers (CD14, CD163). The phenotype of these enriched-in-renal-carcinoma DCs (ercDCs) could be reiterated in vitro by carcinoma-secreted factors (CXCL8/IL-8, IL-6, and vascular endothelial growth factor). ErcDCs resembled conventional DCs in costimulatory molecule expression and antigen cross-presentation. They did not suppress cognate cytotoxic T-lymphocyte function and did not cause CD3ζ down-regulation, FOXP3 induction, or T-cell apoptosis in situ or in vitro; thus, they are different from classic myeloid-derived suppressor cells. ErcDCs secreted high levels of metalloproteinase 9 and used T-cell crosstalk to increase tumor-promoting tumor necrosis factor α and reduce chemokines relevant for T(H)1-polarized lymphocyte recruitment. This modulation of the tumor environment exerted by ercDCs suggests an immunologic mechanism by which tumor control can fail without involving cytotoxic T-lymphocyte inhibition. Pharmacologic targeting of the deviated DC differentiation could improve the efficacy of immunotherapy against RCC.
American Journal Of Pathology 07/2011; 179(1):436-51. · 4.89 Impact Factor
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Sonja Haupt,
Michele Tisdale,
Michelle Vincendeau,
Mary Anne Clements,
David T Gauthier,
Raymond Lance,
O John Semmes,
Adriana Turqueti-Neves, Elfriede Noessner,
Christine Leib-Mösch,
Alex D Greenwood
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ABSTRACT: The human genome comprises approximately 8-9 % of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples.
Journal of General Virology 06/2011; 92(Pt 10):2356-66. · 3.36 Impact Factor
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ABSTRACT: Heat shock proteins (Hsps) hold a dual role depending on their location. Inside cells, they fulfill essential survival functions as molecular chaperones forming complexes with intracellular polypeptides (self or foreign) to help in protein folding, the resolution of protein aggregates and intracellular protein transport. Released from the cell, they act as messengers communicating the cells' interior protein composition to the immune system for initiation of immune responses against intracellular proteins. Here we describe the mechanisms by which Hsp70, the heat-inducible Hsp70 family member, crosstalks with the immune system. Further, we discuss that clinical hyperthermia could be a way to initiate the immunologic activity of Hsp70 by upregulating its expression and facilitating release through local necrosis.
European journal of cell biology 03/2011; 91(1):48-52. · 3.31 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells that act as master regulators of acquired and innate immune responses. While descriptions of cells with dendritic morphology in rodent kidneys date back to the early 1970s, a network of DCs in the mouse kidney has only recently been described. DCs acquire distinct phenotypic and functional characteristics depending on the microenvironment and the disease stages. Concomitantly, their communication with cells of the adaptive immunity might have tissue-protective or tissue-deleterious consequences. This review summarizes results from recent studies on the role of DCs in experimental renal inflammation.
Nephron Experimental Nephrology 01/2011; 119(4):e83-90. · 1.86 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells that act as master regulators of acquired and innate immune responses. Here, we review the available information on their role in human renal inflammation. In the 1980s and early 1990s, major histocompatibility complex class II antigen- (HLA-DR) positive DCs were first described in normal human kidneys and in the interstitium of kidneys from patients with glomerulonephritis. Several DC subtypes were subsequently distinguished based on their expression of CD1c/BDCA-1, CD141/BDCA-3 and CD209/DC-SIGN (in combination with HLA-DR). These cells were almost exclusively found in the tubulointerstitium, with increased numbers seen during glomerulonephritis. It appears that the human renal tubulointerstitium harbors different DC types which allow the collection of both exogenous as well as endogenous antigens. Plasmacytoid DCs have a plasma cell-like morphology and were commonly found within nodular tubulointerstitial infiltrates. Follicular DCs are rarely seen, but show a predominant localization in organized infiltrates. CD207/langerin is a marker for Langerhans cells. Langerin-positive cells have been found in association with the collecting ducts and urothelium. A functional characterization of these subtypes has been hampered by the difficulty of obtaining samples for analysis. However, these studies are clearly required to define the role of DCs and DC subsets in the pathophysiology of renal disease.
Nephron Experimental Nephrology 01/2011; 119(4):e91-8. · 1.86 Impact Factor
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ABSTRACT: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+)Vβ11(+) NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+) subsets.
Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3(+) and CCR5(+) cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4(neg)CD8(neg) (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3(+), CCR4(+), CCR5(+), CCR6(+), CCR7(+), CCR8(+) and CXCR4(+) DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3(+), CCR6(+) and CCR8(+) cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells.
Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens.
PLoS ONE 01/2011; 6(1):e15714. · 4.09 Impact Factor
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ABSTRACT: Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity is a new option for the treatment of cancer. This approach is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T-cell specificities with shared tumor-specific recognition to develop T-cell receptor (TCR)-engineered T lymphocytes for adoptive therapy of RCC.
We established a T-cell clone from TIL that recognized a human leukocyte antigen (HLA)-A2-restricted tumor antigen. The TCR alpha- and beta-chain genes were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes (PBL). A TCR-expressing indicator line (B3Z-TCR53) was established to screen for antigen prevalence in RCC, other malignancies, and normal cell counterparts.
TCR53-engineered PBL recapitulated the specificity of the TIL and showed tumor-specific HLA-A2-restricted effector activities (IFN-gamma, tumor necrosis factor-alpha, interleukin-2, macrophage inflammatory protein-1beta, cytotoxicity). PBL-TCR53 of healthy donors and RCC patients exhibited similar transduction efficiency, expansion, and polyfunctional profile. Using B3Z-TCR53 cells, 130 tumor and normal cells were screened and shared TCR53 peptide: MHC expression was found in >60% of RCC and 25% of tumor lines of other histology, whereas normal tissue cells were not recognized.
To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills the criteria for utilization in TCR gene therapy and advances T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR ligand.
Clinical Cancer Research 04/2010; 16(8):2333-43. · 7.74 Impact Factor
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Elke Bleifuss,
Henriette Bendz,
Birgit Sirch,
Sylvia Thompson,
Anna Brandl,
Valeria Milani,
Michael W. Graner,
Ingo Drexler,
Maria Kuppner,
Emmanuel Katsanis, Elfriede Noessner,
Rolf-Dieter Issels
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ABSTRACT: The goal of immune-based tumor therapies is the activation of immune cells reactive against a broad spectrum of tumor-expressed antigens. Vaccines based on chaperone proteins appear promising as these proteins naturally exist as complexes with various protein fragments including those derived from tumor-associated antigens. Multi-chaperone systems are expected to have highest polyvalency as different chaperones can carry distinct sets of antigenic fragments. A free-solution isoelectric focusing (FS-IEF) technique was established to generate chaperone-rich cell lysates (CRCL). Results from murine systems support the contention that CRCL induce superior anti-tumor responses than single chaperone vaccines. We established an in vitro model for human melanoma to evaluate the capacity of CRCL to transfer endogenously expressed tumor antigens to the cross-presentation pathway of dendritic cells (DC) for antigen-specific T cell stimulation. CRCL prepared from human melanoma lines contained the four major chaperone proteins Hsp/Hsc70, Hsp90, Grp94/gp96 and calreticulin. The chaperones within the melanoma cell-derived CRCL were functionally active in that they enhanced cross-presentation of exogenous peptides mixed into the CRCL preparation. Superior activity was observed for Hsp70-rich CRCL obtained from heat-stressed melanoma cells. Despite the presence of active chaperones, melanoma cell-derived CRCL failed to transfer endogenously expressed melanoma-associated antigens to DC for cross-presentation and cytotoxic T cell (CTL) recognition, even after increasing intracellular protein levels of tumor antigen or chaperones. These findings reveal limitations of the CRCL approach regarding cross-presentation of endogenously expressed melanoma-associated antigens. Yet, CRCL may be utilized as vehicles to enhance the delivery of exogenous antigens for DC-mediated cross-presentation and T cell stimulation.
International Journal of Hyperthermia 07/2009; 24(8):623-637. · 1.92 Impact Factor
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ABSTRACT: The immune response against prostate cancer seems to be inefficient although tumour cells show an over-expression of tumour-associated antigens suggesting that regulatory networks inhibit immune cell function locally. To address this proposition, lymphocytes within prostate cancer-inflicted tissue were analysed for the expression of markers associated with negative regulatory function and exhaustion. Prostate cancer, benign prostatic hyperplasia and healthy prostate tissues were investigated by immunohistology for CD25, FOXP3, PD-1 and B7-H1. We had previously documented that prostate cancer islets are surrounded by clustered accumulations of CD3+ lymphocytes, which lack perforin and interferon-gamma (IFNgamma) expression, thus are apparently quiescent. Here, we report that these clusters contain numerous CD25+ and FOXP3+ cells. These markers are associated with regulatory T cells, and their presence in lymphocyte clusters near prostate cancer regions indicates an environment with negative impact on immune response against cancer cells. Consistent with this hypothesis, cells expressing PD-1 and its ligand B7-H1, which are markers associated with exhaustion of lymphocyte function, were also detected in the lymphocyte clusters. Expression of molecules associated with inhibition and exhaustion of lymphocytes may reflect events contributing to ineffective immune responses against cancer cells.
European journal of cancer (Oxford, England: 1990) 04/2009; 45(9):1664-72. · 4.12 Impact Factor
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ABSTRACT: Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (K(d) = 0.58 microm at 25 degrees C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (K(d) = 32 microm) and melanoma antigen recognized by T cells (MART-1) (K(d) = 2.4 microm). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (K(d) = 0.04 microm) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.
Biological Chemistry 03/2009; 390(4):305-12. · 2.96 Impact Factor
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ABSTRACT: The MAPKs ERK, JNK, and p38 control diverse aspects of the immune response, including regulation of cytotoxin biology in NK cells and CTL. The chemokine CCL5 is coreleased with the cytotoxins, perforin, the granzymes, and granulysin, during the lethal hit administered by cytotoxic CD8+ T cells (CTL). CCL5 expression is up-regulated relatively late in CTL coincident with their functional maturation 3-7 days after activation. Unlike T cells, NK cells have the ability to kill virally infected or transformed cells when directly isolated from the peripheral circulation. In this study, we show that in contrast to T cells, peripheral blood NK cells express CCL5 constitutively. The use of specific inhibitors of the JNK, ERK, and p38 MAPK pathways showed that the JNK pathway controls expression of CCL5 by NK cells. Promoter-reporter assays identified a compact region of the CCL5 promoter responsible for the constitutive transcription of CCL5 by NK cells. EMSA, chromatin immune precipitation, the use of heterologous promoters, and site-directed mutagenesis demonstrated that transcription in NK cells is largely controlled through binding of the transcription factor specificity protein 1 to a region -75 to -56 upstream of the site of transcriptional initiation. Specificity protein 1 expression, and in turn the constitutive expression of CCL5, was found to be controlled through constitutive activation of the JNK/MAPK pathway in peripheral blood NK cells.
The Journal of Immunology 02/2009; 182(2):1011-20. · 5.79 Impact Factor
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ABSTRACT: Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.
Journal of Biological Chemistry 09/2008; 283(39):26477-83. · 4.77 Impact Factor
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ABSTRACT: Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8 degrees C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes.
Cancer Immunology and Immunotherapy 08/2008; 58(3):361-71. · 3.70 Impact Factor
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ABSTRACT: Tumor cells that show downregulation of their tumor-associated antigens (TAAs) may be able to escape immune-mediated elimination. Therefore, efficient vaccine strategies attempt to target multiple TAAs simultaneously. This is easily achieved in dendritic cell (DC)-based vaccines by introducing antigens in the form of RNA. Although insufficient message may hinder adequate expression of individual TAAs when using total-tumor RNA, high amounts of individual RNAs as pools yield DCs presenting high numbers of specific peptide-major histocompatibility complex ligands with epitopes derived from different TAAs. We used the transfer of RNAs encoding the well-defined melanoma TAAs tyrosinase, Melan-A, CDK4mut, gp100, SNRP116mut, and GPNMBmut to characterize DCs at the levels of transfected RNA, expressed protein and peptide-major histocompatibility complex ligand presentation. TAA-encoding RNA was rapidly degraded in the DCs, allowing only a single surge in protein expression shortly after transfection. We compared the functional capacity of DCs transfected with pools of 3 versus 6 RNAs. Whereas functional assays demonstrated a decrease in stimulatory capacity of DCs transfected with a pool of 3 RNAs by only 30% as compared with single RNAs, a 60% loss was seen with 6 RNAs. We conclude that larger RNA pools result in diminished presentation of individual epitopes and suggest that smaller pools of RNA be transfected into separate DC populations which are then pooled to create multiplex vaccines.
Journla of Immunotherapy 02/2008; 31(1):52-62. · 3.27 Impact Factor
