Einar Martin Aandahl

Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens

ABSTRACT Regulatory T (TR) cells maintain tolerance to self-antigens and control immune responses to alloantigens after organ transplantation. Here, we show that CD4+ CD25+ human TR cells suppress virus-specific T-cell responses. Depletion of TR cells from peripheral blood mononuclear cells enhances T-cell responses to cytomegalovirus and human immunodeficiency virus antigens. We propose that chronic viral infections lead to induction of suppressive TR cells that inhibit the antiviral immune response.

FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism

CD4+CD25+ regulatory T (TR) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive TR (TRadapt) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. TRadapt cells produce PGE2 and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE2 receptor-specific antagonists. In resting CD4+CD25− T cells, treatment with PGE2 induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE2 produced by COX-2-positive TRadapt cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients

Cyclic AMP‐dependent protein kinase A (PKA) type I has been established as an acute inhibitor of T cell activation. For this reason, we investigated the possible role of PKA type I in HIV‐induced T cell dysfunction. T cells from HIV‐infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analog than are normal T cells. A PKA type I‐selective antagonist increases the impaired proliferation of T cells from HIV‐infected patients to normal or subnormal levels (up to 2.8‐fold). Follow‐up of patients after initiation of highly active antiretroviral treatment revealed that a majority of patients have a persistent T cell dysfunction that is normalized by incubation of T cells with Rp‐8‐Br‐cAMPS. These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV infection and that PKA type I may be a potential target for immunomodulating therapy.—Aandahl, E. M., Aukrust, P., Skålhegg, B. S., Müller F., Fr⊘land, S. S., Hansson, V., Taskén, K., Protein kinase A type I antagonist restores immune responses of T cells from HIV‐infected patients. FASEB J. 12, 855–862 (1998)

Generation of highly suppressive adaptive CD8+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4+CD25– T cells leads to generation of adaptive CD4+CD25+FOXP3+ regulatory T (TR) 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+ TR 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+ TR cells express prostaglandin E2, IL‐10 and TGF‐β, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4+ TR cells, the CD8+ TR 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+ TR‐mediated suppression, indicating that other mechanism(s) must be employed by these cells.

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

ObjectiveNaturally occurring regulatory T (TR) cells suppress autoreactive T cells whereas adaptive TR cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive TR cells express COX-2 and produce PGE2 that suppress effector T cells in a manner that is reversed by COX-inhibitors.Methods and resultsHere we demonstrate that CRC patients have elevated levels of PGE2 in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ TR cells. Depletion of TR cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity.ConclusionWe suggest that adaptive TR cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE2-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting TR cells and the PGE2–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.

Inhibition of Antigen-Specific T Cell Proliferation and Cytokine Production by Protein Kinase A Type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4+ and CD8+ T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.

The Cyclic AMP-Epac1-Rap1 Pathway Is Dissociated from Regulation of Effector Functions in Monocytes but Acquires Immunoregulatory Function in Mature Macrophages

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2′-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE2 inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-α production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.

Impaired Secretion of IL-10 by T Cells from Patients with Common Variable Immunodeficiency–Involvement of Protein Kinase A Type I

Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4+/CD8+, CD45RA+/RO+, or CD28− T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID.

An Exploratory Trial of Cyclooxygenase Type 2 Inhibitor in HIV-1 Infection: Downregulated Immune Activation and Improved T Cell-Dependent Vaccine Responses

ABSTRACT Chronic HIV infection is characterized by chronic immune activation and dysfunctional T cells with elevated intracellular cyclic AMP (cAMP), which inhibits the T cell activation capability. cAMP may be induced by prostaglandin E2 following lipopolysaccharide (LPS)-induced upregulation of cyclooxygenase type 2 (COX-2) in monocytes due to the elevated LPS levels in patients with chronic HIV infection. This hypothesis was tested using celecoxib, a COX-2 inhibitor, for 12 weeks in HIV-infected patients without antiretroviral treatment in a prospective, open, randomized exploratory trial. Thirty-one patients were randomized in the trial; 27 completed the study, including 13 patients on celecoxib. Celecoxib reduced chronic immune activation in terms of CD38 density on CD8+ T cells (−24%; P = 0.04), IgA levels (P = 0.04), and a combined score for inflammatory markers (P < 0.05). Celecoxib further reduced the inhibitory surface receptor programmed death 1 (PD-1) on CD8+ T cells (P = 0.01), including PD-1 on the HIV Gag-specific subset (P = 0.02), enhanced the number of CD3+ CD4+ CD25+ CD127lo/− Treg or activated cells (P = 0.02), and improved humoral memory recall responses to a T cell-dependent vaccine (P = 0.04). HIV RNA (P = 0.06) and D dimers (P = 0.07) tended to increase in the controls, whereas interleukin-6 (IL-6) possibly decreased in the treatment arm (P = 0.10). In conclusion, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV infection and improved T cell-dependent functions in vivo.