Increased survival from stage IV melanoma associated with fewer regulatory T Cells.

BACKGROUND: Melanoma often elicits a profound immune response, and this response has been exploited by various immune therapies. These immunotherapies ultimately fail, however, and advanced melanoma is uniformly fatal, suggesting the development of an immune escape mechanism. In this study, markers of immune escape including regulatory T cells (T(regs)), dendritic cells (DCs), and TGF-beta were evaluated in 14 Stage IV melanoma patients and correlated with survival. MATERIALS AND METHODS: Peripheral blood mononuclear cells were isolated from Stage IV melanoma patients and analyzed for T(regs) and DCs by flow cytometry using fluorescent CD3, CD4, CD25, Lin, HLA-DR, CD11c, and CD123 antibodies. Serum TGF-beta levels were evaluated by ELISA from these patients. Clinical data were extracted from the patients' medical records. RESULTS: Stage IV melanoma patients with shorter survival (less than 24 mo) had a significantly higher proportion of T(regs) than those with longer survival (15% versus 8%, respectively, P = 0.004). The numbers of DCs and the serum TGF-beta levels were not significantly different in these two groups. There was an inverse relationship between the percentage of T(regs) and survival, although this did not reach statistical significance (r = -0.35, P = 0.22). There was also an inverse relationship between peripheral T(regs) and DCs. When patients were divided into groups of greater than or less than 7% T(regs), the number of total DCs was higher in the patients with fewer T(regs) than in those with more T(regs), but this did not reach statistical significance (16,535 versus 12,126 total DCs/mL, P = 0.52). CONCLUSIONS: In Stage IV melanoma patients, a high percentage of T(regs) appears to be associated with shorter survival. The inverse relationship of the number of DCs and T(regs) in these patients may provide an insight to the origin of this observation.

Melanoma induces immunosuppression by up-regulating FOXP3(+) regulatory T cells.

BACKGROUND: The immune response to melanoma is rarely curative, suggesting the emergence of immunosuppression. FOXP3-expressing regulatory T cells (T(reg) cells) function to suppress immune responses. The objective of this study was to determine if melanoma evades immune surveillance, in part, by inducing T(reg) cells. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated and exposed to melanoma-conditioned media (MCM) or control media for 1 week. The induction of T(reg) cells in these PBMCs was determined by measuring the proportion of CD25(+)FOXP3(+) T cells in all CD4(+) T cells by flow cytometry. FOXP3 expression was determined by mean fluorescence intensity (MFI) and Western blot. Supernatant cytokines were determined by ELISA. RESULTS: Normal PBMCs exposed to MCM revealed higher proportions of T(reg) cells than those exposed to control media after 6 days (3.4% versus 1.3%, respectively, P < 0.02). The expression of FOXP3 in T(reg) cells from PBMCs exposed to MCM increased over time by MFI and Western blot but was not significantly different than those exposed to control media. The level of IL-10 and TGF-beta in supernatants after 6 days growth was higher in MCM than control media, but this did not reach statistical significance. CONCLUSION: Exposure of PBMCs to melanoma results in induction of FOXP3(+) T(reg) cells.

Functional and genomic changes induced by alveolar transmigration in human neutrophils.

Although the accumulation of neutrophils in the lungs and airways is common to many inflammatory lung diseases, including acute lung injury, the alterations that neutrophils undergo as they leave the peripheral circulation and migrate into the lungs have not been well characterized. Human volunteers were exposed to endotoxin by bronchoscopic instillation. The resulting air space neutrophil accumulation and peripheral blood neutrophils were isolated 16 h later, compared with circulating neutrophils isolated before or after to the pulmonary endotoxin exposure, and compared with circulating neutrophils exposed to endotoxin in vitro. Microarray analysis was performed on air space, circulatory, and in vitro endotoxin-stimulated neutrophils. Functional analysis included the determination of neutrophil apoptosis, chemotaxis, release of cytokines and growth factors, and superoxide anion release. Dramatic gene expression differences were apparent between air space and circulating neutrophils: approximately 15% of expressed genes have altered expression levels, including broad increases in inflammatory- and chemotaxis-related genes, as well as antiapoptotic and IKK-activating pathways. Functional analysis of air space compared with circulating neutrophils showed increased superoxide release, diminished apoptosis, decreased IL-8-induced chemotaxis, and a pattern of IL-8, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha release different from either unstimulated or LPS-stimulated circulating neutrophils. Many of these changes are not elicited by in vitro treatment with endotoxin. Limited differences were detected between circulating neutrophils isolated before and 16 h after pulmonary endotoxin instillation. These results suggest that neutrophils sequestered in the lung become fundamentally different from those resident in the circulation, and this difference is distinct from in vitro activation with endotoxin.

Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis.

Recombinant human activated protein C (rhAPC) is a natural anticoagulant with potentially important anti-inflammatory properties. In humans with severe sepsis, rhAPC treatment reduces mortality, but mechanisms responsible have not been well characterized. Accumulation of activated neutrophils in the lungs and other organs during severe infection contributes to sepsis-induced organ dysfunction, including acute inflammatory lung injury. Because neutrophils express an APC receptor, we hypothesized that immunomodulatory effects of rhAPC occur, in part, via modulation of neutrophil responses. To examine this issue, we performed a double-blinded, placebo-controlled study of rhAPC in a human model of endotoxin-induced pulmonary inflammation. Administration of rhAPC significantly reduced leukocyte accumulation to the airspaces, independent of pulmonary cytokine or chemokine release. Neutrophils recovered from bronchoalveolar lavage fluid of volunteers receiving rhAPC demonstrated decreased chemotaxis ex vivo. Decreased neutrophil chemotaxis following exposure to rhAPC was confirmed in vitro. No differences were detected in gene expression, kinase activation, cytokine release, cell survival, or apoptosis of neutrophils recovered in the presence or absence of rhAPC. These studies demonstrate that rhAPC reduces both endotoxin-induced accumulation of leukocytes in the airspaces and neutrophil chemotaxis. These rhAPC-induced effects on neutrophil function may represent a mechanism by which rhAPC improves survival in patients with sepsis.