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Invasive ovarian cancer is associated with poor outcome. The presence of infiltrating regulatory T-cells (Tregs) suppresses protective anti-tumor immune responses, and their accumulation into the tumor microenvironment correlates with reduced survival in ovarian cancer patients. Here, we conducted a detailed characterization of CD4(+) T-cells, CD8(+) T-cells and Treg subsets in the peripheral blood and malignant ascites fluid from seventeen patients with ovarian carcinoma of epithelial origin. Cell distribution, activation status and proliferation status were assessed by multi-color flow cytometry. In ascites fluid, a significant accumulation of CD8(+) cytotoxic T-cells and Tregs was observed compared to peripheral blood. Furthermore, a skewing toward the CD45RA(-) effector/memory compartment was observed in all T-cell subsets in the ascites fluid, but was most pronounced in the Treg population. Regulatory T-cells in the malignant ascites were more activated and had a higher proliferation rate compared to blood-derived cells from the same patient, and their number in ascites was positively correlated with the number of epithelial cells in effusion. In summary, we demonstrate an accumulation of activated CD4(+), CD8(+) and regulatory T-cells in the cancer microenvironment of ovarian carcinoma.

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.