Blockade of surface-bound TGF-ß on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment.

Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-ß (TGF-ß) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell-mediated killing, which depended on surface-bound TGF-ß on the Tregs Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-ß-dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Toward integrative cancer immunotherapy: targeting the tumor microenvironment.

The development of cancer has historically been attributed to genomic alterations of normal host cells. Accordingly, the aim of most traditional cancer therapies has been to destroy the transformed cells themselves. There is now widespread appreciation that the progressive growth and metastatic spread of cancer cells requires the cooperation of normal host cells (endothelial cells, fibroblasts, other mesenchymal cells, and immune cells), both local to, and at sites distant from, the site at which malignant transformation occurs. It is the balance of these cellular interactions that both determines the natural history of the cancer, and influences its response to therapy. This active tumor-host dynamic has stimulated interest in the tumor microenvironment as a key target for both cancer diagnosis and therapy. Recent data has demonstrated both that the presence of CD8⁺ T cells within a tumor is associated with a good prognosis, and that the eradication of all malignantly transformed cells within a tumor requires that the intra-tumoral concentration of cytolytically active CD8⁺ effector T cells remain above a critical concentration until every tumor cell has been killed. These findings have stimulated two initiatives in the field of cancer immunotherapy that focus on the tumor microenvironment. The first is the development of the immune score as part of the routine diagnostic and prognostic evaluation of human cancers, and the second is the development of combinatorial immune-based therapies that reduce tumor-associated immune suppression to unleash pre-existing or therapeutically-induced tumor immunity. In support of these efforts, the Society for the Immunotherapy of Cancer (SITC) is sponsoring a workshop entitled "Focus on the Target: The Tumor Microenvironment" to be held October 24-25, 2012 in Bethesda, Maryland. This meeting should support development of the immune score, and result in a position paper highlighting opportunities for the development of integrative cancer immunotherapies that sculpt the tumor microenvironment to promote definitive tumor rejection.

CD8+ T cell concentration determines their efficiency in killing cognate antigen-expressing syngeneic mammalian cells in vitro and in mouse tissues.

We describe a quantitative model for assessing the cytolytic activity of antigen-specific CD8+ T cells in vitro and in vivo in which the concentration of antigen-specific CD8+ T cells determines the efficiency with which these cells kill cognate antigen-expressing melanoma cells in packed cell pellets, in three-dimensional collagen-fibrin gels in vitro, and in established melanomas in vivo. In combination with a clonogenic assay for melanoma cells, collagen-fibrin gels are 4,500-5,500-fold more sensitive than the packed cell pellet-type assays generally used to measure CD8+ T cell cytolytic activity. An equation previously used to describe neutrophil bactericidal activity in vitro and in vivo also describes antigen-specific CD8+ T cell-mediated cytolysis of cognate antigen-expressing melanoma cells in collagen-fibrin gels in vitro and in transplanted tumors in vivo. We have used this equation to calculate the critical concentration of antigen-specific CD8+ T cells, which is the concentration of these cells required to hold constant the concentration of a growing population of cognate antigen-expressing melanoma cells. It is approximately 3.5x10(5)/ml collagen-fibrin gel in vitro and approximately 3x10(6)/ml or /g melanoma for previously published studies of ex vivo-activated adoptively transferred tumor antigen-specific CD8+ T cell killing of cognate antigen-expressing melanoma cells in established tumors in vivo. The antigen-specific CD8+ T cell concentration required to kill 100% of 2x10(7)/ml cognate antigen-expressing melanoma cells in collagen fibrin gels is >or=10(7)/ml of gel.

Gemfibrozil inhibits Legionella pneumophila and Mycobacterium tuberculosis enoyl coenzyme A reductases and blocks intracellular growth of these bacteria in macrophages.

We report here that gemfibrozil (GFZ) inhibits axenic and intracellular growth of Legionella pneumophila and of 27 strains of wild-type and multidrug-resistant Mycobacterium tuberculosis in bacteriological medium and in human and mouse macrophages, respectively. At a concentration of 0.4 mM, GFZ completely inhibited L. pneumophila fatty acid synthesis, while at 0.12 mM it promoted cytoplasmic accumulation of polyhydroxybutyrate. To assess the mechanism(s) of these effects, we cloned an L. pneumophila FabI enoyl reductase homolog that complemented for growth an Escherichia coli strain carrying a temperature-sensitive enoyl reductase and rendered the complemented E. coli strain sensitive to GFZ at the nonpermissive temperature. GFZ noncompetitively inhibited this L. pneumophila FabI homolog, as well as M. tuberculosis InhA and E. coli FabI.

PAF-receptor antagonists, lovastatin, and the PTK inhibitor genistein inhibit H2O2 secretion by macrophages cultured on oxidized-LDL matrices.

Adhesion of mononuclear phagocytes (Macs) to extracellular matrices containing oxidized low-density lipoproteins (oxLDL) stimulates these cells to secrete reactive oxygen species (e.g., O2-, H2O2) that are believed to promote atherogenesis. Current in vitro systems designed to measure Mac H2O2 secretion in response to oxLDL show that these cells secrete H2O2 for only a few hours after plating. The slow onset and chronicity of atherogenesis, however, suggested to us that Mac ROS secretion might be sustained for much longer periods when Macs are maintained in an environment resembling that in the intima of arteries undergoing atherogenesis. The findings reported here confirm this suggestion. They show that Macs maintained on collagen IV matrices containing oxLDL in medium containing human plasma-derived serum secrete H2O2 continuously and in large amounts for at least 11 days. Using this system we tested the effects of compounds known to attenuate atherogenesis in vivo. Platelet-activating factor (PAF) receptor antagonists, lovastatin, and the isoflavone protein tyrosine kinase (PTK) inhibitor genistein each reduced H2O2 secretion by Macs maintained on oxLDL-containing matrices by approximately 60%. Lovastatin's inhibitory effect was blocked completely by addition of geranylgeranyl pyrophosphate to the medium. We conclude that matrix-bound and oxidized lipoproteins stimulate Macs to produce H2O2 continuously and in large quantities via a pathway that involves PAF receptors and PTK and is reversibly blocked by inhibitors of protein prenylation.

CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.

A pathological hallmark of Alzheimer's disease is the senile plaque, composed of beta-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to beta-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H(2)O(2) secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H(2)O(2) by microglia interacting with fibrillar beta-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar beta-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar beta-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H(2)O(2) production by N9 microglia and human macrophages adherent to fibrillar beta-amyloid by approximately 50%. Our data identify a role for CD36 in fibrillar beta-amyloid-induced H(2)O(2) production by microglia, and imply that CD36 can mediate binding to fibrillar beta-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar beta-amyloid.