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Adrenomedullin (AM) is a novel vasodilatatory peptide which acts primarily through the calcitonin receptor-like receptor (CLR) in combination with either receptor-activity-modifying-protein (RAMP) 2 or 3 (forming receptors, AM(1) and AM(2) respectively). AM plays an important role during inflammation, with its expression increasing following cytokine treatment, promoting macrophage action in situ and high expression by T cells during hypoxic conditions. Examination of T cell AM receptor expression has previously been incomplete, hence we here consider the presentation of AM receptors and their responsiveness to AM and glucocorticoids (GC). AM receptor expression was examined by PCR and flow cytometry in primary human T cells, revealing that RAMP2, 3 and CLR are physiologically expressed in unstimulated T cells, both intracellularly and on the cell surface. PHA stimulation decreased receptor proteins, significantly so for CLR and RAMP3. Incubation with AM elicited limited receptor alterations however, GC treatment (10(-6) M; 24 h) markedly affected cell surface expression, significantly increasing receptor components in unstimulated cells and significantly decreasing the same in stimulated T cells. Our findings indicate that human T cells utilize both AM(1) and AM(2) receptors, which are GC-sensitive in an activation-state dependent manner., (Copyright © 2012 Elsevier B.V. All rights reserved.)

There is an accumulating evidence for the immunoregulatory role of the neuropeptide, nociceptin/orphanin FQ (N/OFQ) however its role on T cell function requires elucidation. This study has demonstrated an inhibitory role for N/OFQ on SEB-activated T cell function. N/OFQ decreases T cell proliferation, which is abrogated when the costimulatory receptors CD80 and CD86 are blocked. In addition, evidence suggests that the immunoregulatory cytokines TGF-beta, IFN-gamma and nitric oxide (NO) are involved in the N/OFQ effect. N/OFQ also, through involvement of IFN and NO, induces the expression of the immunosuppressive modulator indoleamine 2,3-dioxygenase (IDO), suggesting a central role for IDO in the N/OFQ effect on T cell proliferation. The data presented in this report indicate a multi-faceted mechanism of action used by N/OFQ to modulate T cell function.

T-cell clones (TCC) derived from human peripheral blood lymphocytes of a young control, a healthy elderly (SENIEUR) donor, or from CD34(+) hematopoietic progenitor cells were utilised in this study to examine how in vivo and in vitro ageing affects T-cell apoptotic capability. The role of CD25, CD28 and the intracellular proteins, FLICE-inhibitory protein (FLIP), receptor-interacting protein (RIP) and caspase 3 were investigated. We observed an age-related decline in the expression of the IL-2 receptor alpha chain CD25, and absence of the co-stimulatory receptor CD28 on three of the four TCC studied. In young donor- and CD34 cell-derived TCC, but not in SENIEUR donor-derived TCC, we observed an age-related increase in susceptibility of the cells to mFas-L-induced apoptosis, which correlated with the age-related decrease of CD25 expression. Expression levels of full-length RIP and FLIP did not show any correlation to apoptotic susceptibility. However, expression levels of the cleaved form of RIP were greatly reduced in the SENIEUR donor-derived TCC, which together with a trend towards increased caspase 3 activity, could indicate an age-related alteration in utilisation of different apoptotic signalling pathways.

Although nociceptin/orphanin FQ (N/OFQ) and its receptor (ORL-1) are widely distributed throughout the immune system, its role has yet to be elucidated. This study shows that N/OFQ (10(-14)-10(-12) M) modulates T cell activation by up-regulating activation marker expression, e.g. CD28, leading to enhanced proliferation and modulation of TNFalpha secretion. However, on re-stimulated T cells N/OFQ causes inhibition of proliferation, which could be linked with N/OFQ up-regulating CTLA-4 expression. We have also shown that some of these effects are partly prostaglandin-dependent and that N/OFQ induces prostaglandin synthesis. This report suggests that N/OFQ could exert a key modulatory role in human T cell functions.

CD95-induced apoptosis is an important regulatory mechanism in T cells and this complex signalling pathway is now thought to include the protein kinase RIP. Although, RIP is best known for its role in TNF signalling and NF-kappaB activation, it contains a death domain and it is capable of causing apoptosis upon cleavage. In the present study, the role of RIP in CD95-induced apoptosis and its inter-relationship with the caspase cascade was investigated. Studies were performed on both a RIP-/- T cell line and peripheral T lymphocytes, where RIP was degraded through the addition of geldanamycin. Apoptosis was induced by membrane CD95-L, thought to be the most physiological relevant form of CD95-L. Results showed that RIP-/- cells had a decreased susceptibility to death, thus confirming a role for RIP in CD95-induced apoptosis. Furthermore, it was confirmed that RIP is cleaved upon CD95-L stimulation, a process that can be inhibited by Z-VAD. However, only partial inhibition in peripheral T lymphocytes by Z-VAD was observed, suggesting a potential caspase-independent processing of RIP. Studies performed on the activity of effector caspase 3 and on the initiator caspases 2, 8, and 9 revealed that, in the absence of RIP, the activity of these caspases decreases, indicating that RIP-associated apoptosis is caspase-dependent. Hence, these studies support a caspase-related role for RIP in CD95-induced T apoptosis.

P-glycoprotein (Pgp) is a membrane transporter responsible for resistance to chemotherapy in cancer cells. Its presence in T cells is very well documented, but its function in the immune system is still poorly understood. Recent findings suggest that Pgp may be involved in regulating programmed cell death by inhibiting caspase 8 and caspase 3. Utilising antigenically-activated T cells and the physiologically relevant apoptotic ligand, membrane CD95-L, we have previously reported that while T cells are generally resistant to CD95-induced death at early stages of activation, their susceptibility to apoptosis increases with successive activation and clonal expansion. In this study we investigated whether changes in apoptotic susceptibility were related to T cell Pgp function. Results showed that Pgp expression and function in T cells decreases with maturation, with CD8 cells having the highest Pgp function. However, although Pgp function inversely correlated with caspase 3 activity, no difference was observed between apoptotic susceptible CD25- cells and resistant CD25+ cells. In addition sorting of cells with high and low Pgp function showed no correlation with apoptotic capability. Therefore, whilst Pgp modulates caspase activity, it is not responsible for resistance to apoptosis of early activated T cells nor the increased susceptibility observed at the later stages of maturation in antigenically activated cells.

Neuro-inflammation is characterized by immune cell infiltration across the blood-brain barrier, a process instrumental in neuronal cell death. In neuro-inflammation the blood-brain barrier is also damaged and the consequences of activated lymphocytes on the integrity of the blood-brain barrier is not well characterized. Utilizing an blood-brain barrier model we demonstrate that endothelial cell viability and barrier integrity are directly altered following lymphocyte exposure. The effect of activated lymphocytes is cell number dependent, mostly mediated by direct contact, and is not associated with the pro-inflammatory cytokine TNF-alpha. For the successful treatment of neuro-inflammatory disease, intervention of this direct effect at the blood-brain barrier is warranted.

Immunosenescence is believed to contribute to increase susceptibility to infectious diseases and cancer in the elderly, and is caused mainly by changes in the T cell compartment. Longitudinal studies were undertaken to examine T cell surface receptor expression and apoptotic susceptibility using Staphylococcal enterotoxin B (SEB) activated human T cells as an in vitro model of an ageing T cell culture. An intracellular stain Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to assess the number of population divisions (PD) occurring in the ageing T cell culture. One major biomarker of aged T cells is a decrease in expression of CD28 and since this is an essential co-stimulatory molecule, its decreasing expression with age could compromise their activation and apoptotic capacity. Activation of T cells resulted in initial up-regulation of CD25, CD95 and CD28, although expression of CD25 and CD28 subsequently decreased with increasing PD. CD4 and CD8 T cells expressed similar CD25 profiles although CD28 expression was unique in each subset. CD4+ cells expressed the highest CD28 levels, and showed a gradual decline in expression with increasing PD, whereas CD8+ cells were low CD28 expressers, but did not appear to lose their expression as they aged. To determine T cell susceptibility to apoptosis via CD95/CD95-L interactions with increasing age, cells were challenged with CD95-L transfected CHO cells at various PD. Increased death was observed as they aged, which correlated with the decreased expression of activation markers CD25 and CD28.

The ageing immune system shows a gradual decline in responsiveness to antigens and tumours due to the emergence of immunosenescence. The main functions of T cells are activation, anergy and apoptosis and these are all affected during ageing. Apoptosis is vital in controlling cell numbers, deleting self-reactive T cells and maintaining immune surveillance. One of the principle instigators of death involves the CD95:CD95-ligand interaction and as T cells age both receptor and ligand levels increase. This view will describe the current knowledge of the apoptotic susceptibility of ageing T cells and evaluate the factors that may affect the apoptotic capability of immunosenescent T cells.

The generation of effective immunity requires that antigen-specific T cells are activated, clonally expanded and ultimately eliminated by apoptosis. The involvement of CD95-mediated apoptosis in T-cell elimination is well established, but the conditions which regulate the death pathway under normal circumstances are still emerging. Using superantigen-activated human T cells, we found that whilst T-cell receptor (TCR) signalling triggered up-regulation of CD95 ligand (CD95L), the majority of T cells were resistant to apoptosis induction, despite co-expressing high levels of CD95. Resistance was maintained following direct antibody-mediated cross-linking of CD95 and was not confined to early time periods following activation. Our data implicate TCR-derived signals in protection from apoptosis and reveal a role for the mitogen-activated protein (MAP) kinase pathway by use of a MAP kinase kinase (MEK) inhibitor. Collectively these data demonstrate that resistance to activation-induced cell death in human T cells is prolonged rather than transient, is not attributable to a lack of CD95L up-regulation and is due, at least in part, to signalling via the MEK pathway.

Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation.

CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.

Following antigen engagement of the T-cell receptor (TCR), T-cell survival is largely dictated by the provision of additional signals, such as those from costimulatory receptors and cytokine receptors. Whilst CD28-mediated signalling is increasingly associated with survival, ligation of alternative T-cell antigens, such as Fas (CD95), can trigger apoptosis. The T-cell response following antigen engagement may therefore be influenced by the relative expression levels of these coreceptors as well as by the availability of their ligands (CD80/86 and Fas-L). In this study we demonstrate functional interplay between the death receptor Fas and the costimulatory receptor CD28 in human T cells. In Jurkat T cells, we show that Fas signalling leads to rapid and selective CD28 down-regulation, and that this is associated with a specific decrease in mRNA for CD28, indicating that mechanisms exist which target CD28 at a transcriptional level. Moreover, cells that down-regulate CD28 also undergo apoptosis. Studies on activated human peripheral blood T cells demonstrate that cells expressing high levels of CD28 are resistant to Fas-mediated apoptosis whereas cells expressing low levels are more susceptible, implicating CD28 in the provision of anti-apoptotic signals. Consistent with this hypothesis, direct ligation of CD28 using B7 transfectants concomitant with anti-Fas challenge protects from apoptosis. Since antigen-presenting cells may express Fas-L under certain circumstances, the maintenance of T-cell CD28 expression may be crucial for the prevention of Fas-mediated apoptosis during the course of antigen engagement.