TGF-beta modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression.

This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon.

Tumor-induced impairment of TCR signaling results in compromised functionality of tumor-infiltrating regulatory T cells.

This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4(+)CD25(+)FoxP3(+)GITR(high)CD27(low)CD62L(-). The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase.

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells.

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting.

Immune consequences of protracted host-tumor interactions in a transgenic mouse model of mammary carcinoma.

A transgenic mouse model of autochthonous mammary carcinoma was chosen to study the impact of tumor progression on the immune system over an extended period. We found: i) that splenocyte numbers, particularly myeloid cells, increased concurrently with tumor burden; ii) the percentage of tumor-infiltrating Treg cells was similar to that in human breast cancer; iii) suppressed T cell proliferation and cytokine production and; iv) significantly elevated MCP-1 and TNF-alpha in the sera of tumor-bearing mice. The modified immune status in these tumor-bearing hosts is consistent with a "syndrome" that likely impacts the efficacy of cancer immunosurveillance and response to therapy.

Fc-mOX40L fusion protein produces complete remission and enhanced survival in 2 murine tumor models.

OX40L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to CD4+ and CD8+ T cells while inhibiting the effects of suppressive CD4+ CD25+ regulatory T cells. Because of this dual activity, OX40L may provide significant antitumor immunity in tumor-bearing mice. To study its clinical potential, a fusion protein consisting of mOX40L linked to the C-terminus of the Fc fragment of immunoglobulin was genetically engineered.

Construction and preclinical characterization of Fc-mGITRL for the immunotherapy of cancer.

Purpose: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols.

Experimental Design: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor-related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models.

Results: In vivo, construct #178-14 (-5aa, -linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 microg and was found to be intact by gel electrophoresis.

Poly(D,L-lactic-co-glycolic acid) microsphere delivery of adenovirus for vaccination.

Purpose: To study the effect of encapsulation of recombinant adenovirus type 5 encoding Beta-galactosidase (Ad5-Betagal) in poly (D,Llactic-co-glycolic acid) (PLGA) microspheres on viral delivery to professional antigen presenting cells (APCs) in vitro, viral dissemination in vivo, and induction of protective immune responses in vivo.

Methods: PLGA microspheres containing Ad5-alphagal were prepared by a double emulsion solvent evaporation method. Encapsulation efficiency, in vitro release profile, in vitro cellular uptake and in vivo biodistribution of Ad5-alphagal loaded PLGA microspheres were determined using 125I-labeled Ad5-alphagal (125I-Ad5-alphagal).

Immunological responses can have both pro- and antitumour effects: implications for immunotherapy.

Immune responses influence the development and progression of a malignancy. The tumour can also manipulate the immune system to its own ends, often resulting in an ineffective or transient antitumour response. An appreciation of the complexity of these host-tumour interactions is therefore important for the development of more-effective cancer therapies.

Biodegradable nanoparticle delivery of a Th2-biased peptide for induction of Th1 immune responses.

The type of immune response developed against the hepatitis B virus (HBV) is crucial in determining the outcome of the disease. The protective effects of vaccine-induced antibody responses against subsequent exposure to HBV are well-established. After the establishment of chronic HBV infection, cell-mediated immune response is curative while humoral response is detrimental.

Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide.

Regulatory T cells (T(REGs)) control the key aspects of tolerance and play a role in the lack of antitumor immune responses. Cyclophosphamide (CY) is a chemotherapeutic agent with a dose-dependent, bimodal effect on the immune system. Although a previous study demonstrated that CY reduces the number of T(REGs), the mechanism involved in this process has yet to be defined.

Analysis of peptide and lipopeptide content in liposomes.

Purpose: To evaluate several methods for extraction of peptides from liposomal formulations as a first step in their quantification, and to determine the encapsulation efficiency for a panel of 8 peptides.

Methods: Eight peptides were chosen due to their importance in the field of vaccine development. Three different extraction media were examined: 25% ethanol, 98% ethanol, and 100% methanol.

Analysis of poly(D,L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro.

Purpose: The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs).

Methods: Parallel cultures of DCs and macrophages (Mphi) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for Mphi). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique.