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-b, 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. IntroductionCD4 + regulatory T (T R ) cells maintain self-tolerance to autoantigens and are involved in the pathogenesis of various clinical conditions such as autoimmune diseases, chronic viral infections and cancer. Several subsets of CD4 + T R cells have been characterized [1,2]. Whereas naturally occurring CD4 + CD25 + FOXP3 + T R cells are generated in the thymus and suppress effector T cells in a cell contact-dependent manner [3][4][5], adaptive CD4 + CD25 + FOXP3 + T R cells are induced from naive T cells in the periphery and the suppressive activity is independent of cell contact [2,[6][7][8].Suppressive T cells are not strictly confined to the CD4 + T cell compartment and CD8 + T R cells have been characterized in both clinical and experimental conditions [9][10][11][12]. The CD8 + T R cells share phenotypic features with CD4 + T R cells, and as their counterpart, CD8 + T R cells can be generated both in the thymus and in the periphery [13][14][15][16][17][18].In humans, two subsets of adaptive CD8 + CD28 -T R cells exist. Type 1 CD8 + T R cells are generated by stimulation of naive T cells with allogeneic antigenpresenting cells (APC) [19][20][21] properties appears in parallel with up-regulation and expression of cytotoxic molecules. Although the adaptive CD8 + T R cells express prostaglandin E 2 (PGE 2 ), IL-10 and TGF-b, the suppressive mechanism appears to be cell contact-dependent. This is in contrast to adaptive CD4 + T R cells that inhibit T cell immune responses by secretion of humoral factors. Figure 1. Generation of adaptive CD8 + T R cells by continuous antigen stimulation. (A) CD25 + cell-depleted PBMC were stimulated with SEB (4 days). CD8 + CD25 + T cells were added in increasing concentrations to CD25 -cells stimulated with SEB from the autologous blood donor. Proliferation of responding T cells was assessed by CFSE proliferation assay. Representative data are shown (n = 2...

We suggest that adaptive T(R) cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2-PGE(2)-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting T(R) cells and the PGE(2)-cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

Monocytes initiate innate immune responses and interact with T cells to induce antigen-specific immune responses by antigen presentation and secretion of humoral factors. We have previously shown that adaptive regulatory T cells inhibit T-cell effector functions in a cyclooxygenase (COX)-2-prostaglandin E(2) (PGE(2))-dependent manner and that PGE(2) converts resting CD4+CD25- T cells into FOXP3+ T cells with a suppressive phenotype. Here, we demonstrate that stimulation of monocytes with LPS leads to suppression of T-cell immune responses by a COX-2-PGE(2)-dependent mechanism that is reversible with COX-2 inhibitors as well as PGE(2)-neutralizing antibody and cAMP antagonist. Furthermore, we show that LPS-activated monocytes induce FOXP3 expression in resting CD4+CD25- T cells by the same pathway. These results suggest that monocytes are able to efficiently suppress T-cell immune responses in a regulatory manner and elicit an inhibitory immune profile.

Adaptive regulatory T cells (Tregs) contribute to an immunosuppressive microenvironment in colorectal cancer (CRC). Here, we examined whether the level of Treg-mediated inhibition of antitumor immune responses in patients with metastatic CRC (metCRC) selected for liver resection is associated with clinical outcome. Preoperatively and at follow-ups, we did flow-based phenotyping, examined antitumor immunity using peptides from carcinoembryonic antigen (CEA) protein in the presence or absence of CD4(+)CD25(+)CD127(dim/-) cells (Tregs) and determined cytokine and PGE(2) levels in patient blood samples. At 18 months post-surgery, 8 patients were disease free (7 alive and 1 dead of unrelated cause) and 10 had experienced disease recurrence (7 alive and 3 dead of metCRC). Prior to surgery, the patients demonstrated Treg-mediated suppression of TNFα and IFNγ expression that could be perturbed through the PGE(2)/cAMP pathway and the immune suppression was significantly higher in the group that later developed disease recurrence (P = 0.046). Furthermore, the post-surgery plasma PGE(2) levels were related to the clinical outcome (PGE(2) levels of 280 ± 47 vs. 704 ± 153 pg/ml (mean ± SEM) for disease free and recurrent disease, respectively). T-cell phenotyping revealed higher frequencies of COX-2(+) cells in the patients with recurrent disease. These findings support the notion that the level of Treg-mediated suppression of adaptive antitumor immune responses at the time of surgery may influence later clinical outcome of metCRC and provide valuable prognostic information.

Human CD4+CD25+ regulatory T (T(R)) cells express the transcription factor forkhead box p3 (FOXP3) and have potent immunosuppressive properties. While naturally occurring T(R) cells develop in the thymus, adaptive T(R) cells develop in the periphery from naive CD4+ T cells. Adaptive T(R) cells may express cyclooxygenase type 2 (COX-2) and suppress effector T cells by a PGE(2)-dependent mechanism, which is reversible with COX inhibitors. In this study we have characterized the differentiation of naive CD4+ T cells into adaptive T(R) cells in detail during 7 days of continuous antigen stimulation. After 2 days of stimulation of CD4+CD25- T cells, the cells expressed FOXP3 and COX-2 and displayed potent immunosuppressive properties. The suppressive phenotype was present at all observed time-points from Day 2, although suppression was merely present at Day 7. The adaptive T(R) cells expressed cell surface markers consistent with an activated phenotype and secreted high levels of TGF-beta, IL-10, and PGE(2). However, the suppressive phenotype was found exclusively in cells that proliferated upon activation. These data support the notion that activation of naive CD4+ T cells leads to concomitant acquisition of effector and suppressive properties.

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However, the activation requirements, the temporal aspects of the suppressive activity, and mode of action of human Tregs are subjects of controversy. In this study, we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously, whereas in the remaining donors, anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore, anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs, but could be fully obliterated by trypsin treatment, indicating that a cell surface protein is directly involved. By add-back of active, fixed Tregs at different time points after activation of responding T cells, the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly, we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation, indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells.

Patients with colorectal cancer (CRC) have been shown to have elevated levels of circulating prostaglandin E (PGE) which promotes cancer progression and suppresses T cell immune responses. In this study we evaluated whether signaling responses in T lymphocytes obtained from peripheral blood of CRC patients were affected by the sustained exposure to increased levels of PGE. The phosphorylation status of an extended panel of proteins involved in downstream signaling cascades in T cells was profiled at a single cell level both in naïve and antigen-experienced cells after triggering T cell-, prostaglandin- and interleukin-2 receptors. Peripheral T cells from patients with elevated PGE levels displayed aberrant T cell signaling responses downstream of the T cell receptor (assessed by reduced phosphorylation of CD3ζ and SLP76), and after triggering the IL-2 receptor (assessed by reduced phosphorylation of STAT5) when compared to T cells from CRC patients with lower levels of PGE and T cells from healthy blood donors. This signaling study of circulating T cells from CRC patients indicates that increased systemic PGE levels affect proximal T cell responses and confirms phospho-specific flow cytometry to be a valuable tool for revealing signaling signatures in immunological disorders.

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