Will telomere erosion lead to a loss of T-cell memory?

Evidence is accumulating that elderly individuals are more susceptible to infection with organisms to which they were previously immune. This indicates that there might be a limit to the persistence of immune memory. This fact is particularly disturbing because the average life expectancy of humans has almost doubled in the past 200 years and is still increasing. We discuss mechanisms that might constrain the persistence of memory T cells and consider whether humans will suffer from memory T-cell exhaustion as life expectancy increases.

Endogenous cortisol and TGF-beta in human aqueous humor contribute to ocular immune privilege by regulating dendritic cell function.

Aqueous humor (AqH) has been shown to have significant immunosuppressive effects on APCs in animal models. We wanted to establish whether, in humans, AqH can regulate dendritic cell (DC) function and to identify the dominant mechanism involved. Human AqH inhibited the capacity of human peripheral blood monocyte-derived DC to induce naive CD4(+) T cell proliferation and cytokine production in vitro, associated with a reduction in DC expression of the costimulatory molecule CD86. This was seen both for DC cultured under noninflammatory conditions (immature DC) and for DC stimulated by proinflammatory cytokines (mature DC). DC expression of MHC classes I/II and CD83 was reduced (mature DC only). Myeloid DC from peripheral blood were similarly sensitive to the effects of human AqH, but only under inflammatory conditions. The addition of α-melanocyte stimulating hormone and vasoactive intestinal peptide did not cause significant inhibition at physiological levels. However, the addition of exogenous cortisol at physiological levels recapitulated the AqH-induced reduction in CD86 and inhibition of DC-induced T cell proliferation, and blockade of cortisol in AqH partially reversed its suppressive effects. TGF-ß2 had an additional effect with cortisol, and although simultaneous blockade of cortisol and TGF-ß2 in AqH reduced its effectiveness, there was still a cortisol- and TGF-ß-independent component. In humans, AqH regulates DC maturation and function by the combined actions of cortisol and TGF-ß2, a pathway that is likely to contribute to the maintenance of immune privilege in the eye.

Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO.

Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with previous reports that the ( approximately 80%) majority of natural Tregs in adults are CD45RO(+). The proportion of CD45RA(+) Tregs decreases, whereas CD45RO(+) Tregs increase significantly with age. Nevertheless, a small proportion of CD45RA(+) Tregs are found even in old (>80 y) adults and a proportion of these express CD31, a marker for recent thymic emigrants. We found that CD45RO(+) Tregs were highly proliferative compared with their CD45RA(+) counterparts. This was due in part to the conversion of CD45RA Tregs to CD45RO expression after activation. Another difference between these two Treg populations was their preferential migration to different tissues in vivo. Whereas CD45RA(+) Tregs were preferentially located in the bone marrow, associated with increased CXCR4 expression, CD45RO(+) Tregs were preferentially located in the skin, and this was associated with their increased expression of CLA and CCR4. Our studies therefore show that proliferation features strongly in maintenance of the adult Treg pool in humans and that the thymus may make a minor contribution to the maintenance of the peripheral pool of these cells, even in older adults. Furthermore, the different tissue compartmentalization of these cells suggests that different Treg niches exist in vivo, which may have important roles for their maturation and function.

Novel antileukemic compound ingenol 3-angelate inhibits T cell apoptosis by activating protein kinase Ctheta.

Members of the protein kinase C (PKC) family of serine-threonine kinases are important regulators of immune cell survival. Ingenol 3-angelate (PEP005) activates a broad range of PKC isoforms and induces apoptosis in acute myeloid leukemia cells by activating the PKC isoform PKCdelta. We show here that, in contrast to its effect on leukemic cells, PEP005 provides a strong survival signal to resting and activated human T cells. The antiapoptotic effect depends upon the activation of PKC. This PKC isoform is expressed in T cells but is absent in myeloid cells. Further studies of the mechanism involved in this process showed that PEP005 inhibited activated CD8(+) T cell apoptosis through the activation of NFkappaB downstream of PKC, leading to increased expression of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Transfection of CD8(+) T cells with dominant-negative PKC diminished the prosurvival effect of PEP005 significantly. Ectopic expression of PKC in the acute myeloid leukemia cell line NB4 turned their response to PEP005 from an increased to decreased rate of apoptosis. Therefore, in contrast to myeloid leukemia cells, PEP005 provides a strong survival signal to T cells, and the expression of functional PKC influences whether PKC activation leads to an anti- or proapoptotic outcome in the cell types tested.

The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo.

Naturally occurring CD4(+)CD25(hi)Foxp3(+) Tregs (nTregs) are highly proliferative in blood. However, the kinetics of their accumulation and proliferation during a localized antigen-specific T cell response is currently unknown. To explore this, we used a human experimental system whereby tuberculin purified protein derivative (PPD) was injected into the skin and the local T cell response analyzed over time. The numbers of both CD4(+)Foxp3(-) (memory) and CD4(+)Foxp3(+) (putative nTreg) T cells increased in parallel, with the 2 populations proliferating at the same relative rate. In contrast to CD4(+)Foxp3(-) T cell populations, skin CD4(+)Foxp3(+) T cells expressed typical Treg markers (i.e., they were CD25(hi), CD127(lo), CD27(+), and CD39(+)) and did not synthesize IL-2 or IFN-gamma after restimulation in vitro, indicating that they were not recently activated effector cells. To determine whether CD4(+)Foxp3(+) T cells in skin could be induced from memory CD4(+) T cells, we expanded skin-derived memory CD4(+) T cells in vitro and anergized them. These cells expressed high levels of CD25 and Foxp3 and suppressed the proliferation of skin-derived responder T cells to PPD challenge. Our data therefore demonstrate that memory and CD4(+) Treg populations are regulated in tandem during a secondary antigenic response. Furthermore, it is possible to isolate effector CD4(+) T cell populations from inflamed tissues and manipulate them to generate Tregs with the potential to suppress inflammatory responses.

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis.

OBJECTIVES: In rheumatoid arthritis (RA), a complex cytokine network drives chronic inflammation and joint destruction. So far, few attempts have been made to identify the cellular sources of individual cytokines systematically. Therefore, the primary objective of this study was systematically to assess the cytokine messenger RNA expression profiles in the five largest cell populations in the synovial fluid and peripheral blood of RA patients. To reflect the in vivo situation as closely as possible, the cells were neither cultured nor stimulated ex vivo. METHODS: Inflammatory cells from 12 RA patients were sorted into CD4 and CD8 T cells, B cells, macrophages and neutrophils. mRNA expression for 41 cytokines was determined by real-time PCR using microfluidic cards. Receptor activator nuclear factor kappa B ligand (RANKL) (TNFSF11) expression by B cells was further confirmed by flow cytometry and by immunofluorescence staining of frozen sections of synovial tissue from patients with RA. RESULTS: The detection of cytokines characteristic for T cells and myeloid cells in the expected populations validated this methodology. Beyond the expected cytokine patterns, novel observations were made. Striking among these was the high expression of mRNA for RANKL in B cells from synovial fluid. This observation was validated at the protein level in synovial tissue and fluid. CONCLUSIONS: RANKL, the key cytokine driving bone destruction by osteoclast activation, is produced by synovial B cells in RA. This observation is of importance for our understanding of the role of B cells in RA and their therapeutic targeting.

Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease characterized by progressive bile duct destruction, develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman, R.W. 1991. Gut. 32:1433-1435). However, the liver and bowel inflammation are rarely concomitant, and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut, but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant, A.J., P.F. Lalor, M. Salmi, S. Jalkanen, and D.H. Adams. 2002. Lancet. 359:150-157). In support of this, we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD.

Telomere erosion in memory T cells induced by telomerase inhibition at the site of antigenic challenge in vivo.

The extent of human memory T cell proliferation, differentiation, and telomere erosion that occurs after a single episode of immune challenge in vivo is unclear. To investigate this, we injected tuberculin purified protein derivative (PPD) into the skin of immune individuals and isolated responsive T cells from the site of antigenic challenge at different times. PPD-specific CD4+ T cells proliferated and differentiated extensively in the skin during this secondary response. Furthermore, significant telomere erosion occurred in specific T cells that respond in the skin, but not in those that are found in the blood from the same individuals. Tissue fluid obtained from the site of PPD challenge in the skin inhibited the induction of the enzyme telomerase in T cells in vitro. Antibody inhibition studies indicated that type I interferon (IFN), which was identified at high levels in the tissue fluid and by immunohistology, was responsible in part for the telomerase inhibition. Furthermore, the addition of IFN-alpha to PPD-stimulated CD4+ T cells directly inhibited telomerase activity in vitro. Therefore, these results suggest that the rate of telomere erosion in proliferating, antigen-specific CD4+ T cells may be accelerated by type I IFN during a secondary response in vivo.

Inflammatory responses of endothelial cells experiencing reduction in flow after conditioning by shear stress.

Exposure of endothelial cells (EC) to shear stress reduces their response to tumour necrosis factor-alpha (TNF). We tested how shear-conditioned EC responded to reduction in flow, either by spontaneously binding leukocytes, or by increasing sensitivity to TNF. Human umbilical vein EC were exposed to shear stress of 2.0 Pa (20 dyn/cm(2)) for 24 h. Shear was then reduced to stasis (30 sec perfusion each hour to exchange medium) or 0.003 Pa (creeping flow). At chosen times, neutrophils were perfused over the EC at 0.1 Pa (effective reperfusion). EC developed an ability to capture flowing neutrophils that lasted from 1 to 3 h after flow reduction, which was reduced by antibody against P-selectin or pre-treatment of EC with an inhibitor of NADPH-oxidase. Adhesion of neutrophils to TNF-treated EC was greatly suppressed by shear-conditioning, remained suppressed immediately after cessation of flow and then took 48 h to approach the level in static cultures. Interestingly, the response to TNF remained suppressed in cultures switched to creeping flow. Gene array analysis confirmed that differently recovered cells had separate phenotypes. Thus, an acute response of EC to reduction in shear may contribute to leukocyte recruitment, along with hypoxia, in ischaemia and reperfusion. Prolonged cessation of flow may increase the sensitivity of EC to inflammatory stimuli, but this effect may be suppressed by residual flow.

The role of chemokines in leucocyte-stromal interactions in rheumatoid arthritis.

New dimensions in our understanding of immune cell trafficking in health and disease have been opened by the discovery of chemokines and their receptors. This family of chemo-attractant cytokines performs essential roles in the recruitment and subsequent positioning of leucocyte subsets within tissue microenvironments. Investigation of chemokine networks offers a novel approach to understand the mechanisms by which inflammatory cells persist in diseases such as rheumatoid arthritis (RA), where evidence is mounting that the inappropriate temporal and spatial expression of chemokines and/or their receptors may impair the resolution of leucocyte infiltrates. The recognition that stromal cells such as fibroblasts, as active components of tissue specific microenvironments, are able to determine the type and persistence of inflammatory infiltrates has opened new vistas in research. Stromal cells are active contributors to cytokine and inflammatory chemokine networks which result in immune cell recruitment and activation. However an intriguing role of stromal cells has been demonstrated in the inappropriate expression of constitutive, housekeeping chemokines, which contribute to the persistence of inflammation by actively blocking its resolution.

CD248/Endosialin is dynamically expressed on a subset of stromal cells during lymphoid tissue development, splenic remodeling and repair.

Studies of stromal cell populations in lymphoid tissue (LT) have been hampered by a lack of selective markers. Here, we show that CD248 (Endosialin/TEM1) is a stromal marker that is differentially expressed on fibroblasts and pericytes in the thymus, lymph node and spleen. Expression is high during LT development but largely disappears in the adult. CD248 is re-expressed in a Salmonella-induced model of splenic enlargement; peak expression corresponding to the peak of splenic enlargement. These results suggest that CD248 expression helps define a subset of LT stromal cells which play a role in remodelling during tissue development, infection and repair.

Identification of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration.

Recent studies have demonstrated that neutrophils are not a homogenous population of cells. Here, we have identified a subset of human neutrophils with a distinct profile of cell-surface receptors [CD54(high), CXC chemokine receptor 1(low) (CXCR1(low))], which represent cells that have migrated through an endothelial monolayer and then re-emerged by reverse transmigration (RT). RT neutrophils, when in contact with endothelium, were rescued from apoptosis, demonstrate functional priming, and were rheologically distinct from neutrophils that had not undergone transendothelial migration. In vivo, 1-2% of peripheral blood neutrophils in patients with systemic inflammation exhibit a RT phenotype. A smaller population existed in healthy donors ( approximately 0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54(low), CXCR1(high)) and naïve cells after activation with formyl-Met-Leu-Phe (CD54(low), CXCR1(low)). It is important that the RT phenotype (CD54(high), CXCR1(low)) is also distinct from tissue-resident neutrophils (CD54(low), CXCR1(low)). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue-experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.

The assessment of T-cell apoptosis in synovial fluid.

T-cell apoptosis is central to the resolution of chronic inflammation. Inhibition of this process of programmed cell death contributes to disease persistence in conditions such as rheumatoid arthritis. An understanding of T-cell apoptosis and its regulation is clearly important for understanding the pathophysiology of inflammatory disease. This chapter describes a number of apoptosis assays that can be used to measure T-cell apoptosis in synovial fluid. The choice of assay depends, in part, on the phase of apoptosis under investigation and this review puts this into context by introducing these phases and their regulation.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments.

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples.

Treating very early rheumatoid arthritis.

Rheumatoid arthritis (RA) is common and leads to joint damage due to persistent synovitis. The persistence of inflammation is maintained by hyperplastic stromal tissue, which drives the accumulation of leukocytes in the synovium. Aggressive treatment after the first 3-4 months of symptoms, with either disease modifying anti-rheumatic drugs or anti-tumor necrosis factor (TNF)-alpha therapy, reduces the rate of disease progression. However, it rarely switches off disease such that remission can be maintained without the continued need for immunosuppressive therapy. There is increasing evidence that the first few months after symptom onset represent a pathologically distinct phase of disease. This very early phase may translate into a therapeutic window of opportunity during which it may be possible to permanently switch off the disease process. The rationale for, and approaches to, treatment within this very early window are discussed.

The impact of telomere erosion on memory CD8+ T cells in patients with X-linked lymphoproliferative syndrome.

Patients with X-linked lymphoproliferative syndrome (XLP) experience excessive T cell proliferation after primary Epstein-Barr virus (EBV) infection, due to mutations in the signalling lymphocyte activation molecule (SLAM) associated protein (SAP) molecule. We examined the impact of dysfunctional proliferative control on the extent of CD8+ T cell differentiation in XLP patients who recovered from primary EBV infection. Although these young patients have normal numbers of lytic and latent EBV-epitope-specific CD8+ T cells, they were extremely differentiated as defined by loss of CCR7 and CD27, low telomerase activity and very short telomeres. This was not a direct effect arising from the loss of SAP, but was due to excessive T cell stimulation due to this defect. Thus, transduction of XLP CD8+ T cells with the catalytic component of telomerase (hTERT), but not SAP, prevented telomere loss and considerably extended proliferative lifespan in vitro. These results indicate that excessive proliferation in CD8+ T cells in XLP patients may lead to end-stage differentiation and loss of functional EBV-specific CD8+ T cells through replicative senescence. This may contribute to the defective immunity found in XLP patients who survive acute EBV infection who develop EBV-related B cell lymphomas before the fourth decade of life.

Generation and characterization of novel stromal specific antibodies.

Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic. From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2) broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

Multiplex bead immunoassay analysis of aqueous humor reveals distinct cytokine profiles in uveitis.

PURPOSE: To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate. METHODS: AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Behçet's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample. RESULTS: Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2. CONCLUSIONS: Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.

The role of chemokines and their receptors in ocular disease.

The migration and infiltration of cells into the eye whether blood-borne leucocytes, endothelial or epithelial cells occurs in many ocular diseases. Dysregulation of this process is apparent in chronic inflammation, corneal graft rejection, allergic eye disease and other sight-threatening conditions. Under normal and inflammatory conditions, chemokines and their receptors are important contributors to cell migration. To date, 47 chemokines and 19 chemokine receptors have been identified and characterised. In recent years, investigations into the role of chemokines and their receptors in ocular disease have generated an increasing number of publications. In the eye, the best understood action of these molecules has arisen from the study of their ability to control the infiltration of leucocytes in uveitis. However, the involvement of chemokines in angiogenesis in several ocular conditions and in the survival of corneal transplants demonstrates the multifaceted nature of their effects. Interestingly, the constitutive expression of chemokines and their receptors in ocular tissues suggests that certain chemokines have a homeostatic function. In this review, we discuss the nature and function of chemokines in health and disease, and describe the role of chemokines in the pathogenesis of different ocular conditions.

The role of leukocyte-stromal interactions in chronic inflammatory joint disease.

Rheumatoid arthritis (RA) is a debilitating, chronic, persistent inflammatory disease that is characterised by painful and swollen joints. The aetiology of RA is unknown, however whereas past research has concentrated on the role of immune or inflammatory infiltrating cells in inflammation, it is becoming clear that stromal cells play a critical part in regulating the quality and duration of an inflammatory response. In this review we assess the role of fibroblasts within the inflamed synovium in modulating immune responses; in particular we examine the role of stromal cells in the switch from resolving to persistent inflammation as is found in the rheumatoid synovium.