Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.

Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations.

Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.

Humanized Mice Reveal New Insights Into the Thymic Selection of Human Autoreactive CD8+ T Cells.

Thymic selection constitutes the first checkpoint in T-cell development to purge autoreactive T cells. Most of our understanding of this process comes from animal models because of the challenges of studying thymopoiesis and how T cell receptor (TCR) specificity impacts thymocyte phenotype in humans. We developed a humanized mouse model involving the introduction of autoreactive TCRs and cognate autoantigens that enables the analysis of selection of human T cells in human thymic tissue in vivo. Here, we describe the thymic development of MART1-specific autoreactive CD8+ T cells that normally escape deletion and how their phenotype and survival are affected by introduction of the missing epitope in the hematopoietic lineage. Expression of the epitope in a fraction of hematopoietic cells, including all major types of antigen-presenting cells (APCs), led to profound yet incomplete deletion of these T cells. Upregulation of PD-1 upon antigen encounter occurred through the different stages of thymocyte development. PD-1 and CCR7 expression were mutually exclusive in both transgenic and non-transgenic thymocytes, challenging the view that CCR7 is necessary for negative selection in humans. In the presence of antigen, MART1-reactive T cells down-regulated TCR, CD3, CD8, and CD4 in the thymus and periphery. Moreover, expression of secondary TCRs influences MHC class I-restricted T cells to develop as CD4+, particularly regulatory T cells. This new model constitutes a valuable tool to better understand the development of autoreactive T cells identified in different human autoimmune diseases and the role of different APC subsets in their selection.

Altered Function of Antigen-Presenting Cells in Type 1 Diabetes: A Challenge for Antigen-Specific Immunotherapy?

Type 1 diabetes (T1D) arises from a failure to maintain tolerance to specific ß-cell antigens. Antigen-specific immunotherapy (ASIT) aims to reestablish immune tolerance through the supply of pertinent antigens to specific cell types or environments that are suitable for eliciting tolerogenic responses. However, antigen-presenting cells (APCs) in T1D patients and in animal models of T1D are affected by a number of alterations, some due to genetic polymorphism. Combination of these alterations, impacting the number, phenotype, and function of APC subsets, may account for both the underlying tolerance deficiency and for the limited efficacy of ASITs so far. In this comprehensive review, we examine different aspects of APC function that are pertinent to tolerance induction and summarize how they are altered in the context of T1D. We attempt to reconcile 25 years of studies on this topic, highlighting genetic, phenotypic, and functional features that are common or distinct between humans and animal models. Finally, we discuss the implications of these defects and the challenges they might pose for the use of ASITs to treat T1D. Better understanding of these APC alterations will help us design more efficient ways to induce tolerance.

Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells.

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28- T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3'UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3'UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3'UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

Age-Associated Differences in MiRNA Signatures Are Restricted to CD45RO Negative T Cells and Are Associated with Changes in the Cellular Composition, Activation and Cellular Ageing.

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.

T-cell Activation Induces Dynamic Changes in miRNA Expression Patterns in CD4 and CD8 T-cell Subsets.

T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T-cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T cells in response to antigen priming.

Low-affinity TCR engagement drives IL-2-dependent post-thymic maintenance of naive CD4+ T cells in aged humans.

Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA(+) CD25(dim) CD4(+) T cells can develop from conventional naive CD25(-) CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA(+) CD25(dim) CD4+ T cells expressed a broad TCRVß repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA(+) CD25(dim) CD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.

Immuno-miRs: critical regulators of T-cell development, function and ageing.

MicroRNAs (miRNAs) are instrumental to many aspects of immunity, including various levels of T-cell immunity. Over the last decade, crucial immune functions were shown to be regulated by specific miRNAs. These 'immuno-miRs' regulate generic cell biological processes in T cells, such as proliferation and apoptosis, as well as a number of T-cell-specific features that are fundamental to the development, differentiation and function of T cells. In this review, we give an overview of the current literature with respect to the role of miRNAs at various stages of T-cell development, maturation, differentiation, activation and ageing. Little is known about the involvement of miRNAs in thymic T-cell development, although miR-181a and miR-150 have been implicated herein. In contrast, several broadly expressed miRNAs including miR-21, miR-155 and miR-17~92, have now been shown to regulate T-cell activation. Other miRNAs, including miR-146a, show a more T-cell-subset-specific expression pattern and are involved in the regulation of processes unique to that specific T-cell subset. Importantly, differences in the miRNA target gene repertoires of different T-cell subsets allow similar miRNAs to control different T-cell-subset-specific functions. Interestingly, several of the here described immuno-miRs have also been implicated in T-cell ageing and there are clear indications for causal involvement of miRNAs in immunosenescence. It is concluded that immuno-miRs have a dynamic regulatory role in many aspects of T-cell differentiation, activation, function and ageing. An important notion when studying miRNAs in relation to T-cell biology is that specific immuno-miRs may have quite unrelated functions in closely related T-cell subsets.

Targeted folate receptor ß fluorescence imaging as a measure of inflammation to estimate vulnerability within human atherosclerotic carotid plaque.

UNLABELLED: The probability of atherosclerotic plaque rupture and its thrombotic sequelae are thought to be increased at sites of macrophage accumulation. Folate receptor ß (FR-ß) is present on activated macrophages but not on quiescent macrophages or other immune cells. By conjugating the ligand folate with a fluorescent contrast agent, fluorescein isothiocyanate (FITC), we aimed to explore the potential role of FR-ß fluorescence imaging in the distinction of vulnerable sites from more stable regions. METHODS: Carotid specimens were taken from 20 patients and incubated with folate-FITC for 30 min. Ex vivo fluorescence imaging was performed to determine the exact location of folate-FITC uptake. Sections displaying regions of high uptake (determined as hot spots) were compared with sections showing low uptake (cold spots) through immunohistochemistry and real-time quantitative reverse-transcription polymerase chain reaction for FR-ß. RESULTS: Hot spots showed significantly higher folate-FITC uptake than cold spots (P < 0.001). Hot spots tended to contain more macrophages and areas of hypoxia than cold spots. A positive correlation between messenger RNA levels of CD68 (marker for macrophages), FR-ß (r = 0.53, P = 0.045), and hypoxia-inducible factor-1α expression (marker for intraplaque hypoxia; r = 0.55, P = 0.034) was found. CONCLUSION: Compared with areas with low folate-FITC uptake, areas of high folate-FITC uptake within human atherosclerotic plaques had an increased number of activated macrophages and higher areas of hypoxia. These characteristics of vulnerability imply that molecular imaging of FR-ß through folate conjugates might be a good indicator for plaque vulnerability in future noninvasive imaging studies.

Macrophage folate receptor-ß (FR-ß) expression in auto-immune inflammatory rheumatic diseases: a forthcoming marker for cardiovascular risk?

In patients with systemic auto-immune inflammatory rheumatic diseases (AIIRD) like rheumatoid arthritis the prevalence of cardiovascular disease (CVD) is increased. In the pathogenesis of AIIRD and atherosclerosis many similarities can be found in the process underlying CVD. Accumulation of inflammatory cells, in particular macrophages at the site of inflammation producing inflammatory mediators serve as a prominent feature in both systemic inflammation and atherosclerosis. Two different subtypes of macrophages have been described in recent literature namely classically activated macrophages (M1) and alternatively activated macrophages (M2). Alternatively activated macrophages are characterized by low CD14 and high CD163 expression. Macrophages expressing CD14 (M1) have been identified within atherosclerotic plaques, whereas CD14 low macrophages are abundant in vessels without atherosclerosis. Depending on the environment and responses to different stimuli, macrophages in plaques can express diverse pro and anti-atherogenic functions. The balance of these different activation profiles influences atheroma evolution and outcome. Nowadays, influx of macrophages is recognized as a very important feature of the pathogenesis of plaque formation. Activated macrophages accumulate at the sites of inflammation and can therefore be exploited to better visualize inflammatory responses in atherosclerosis. Furthermore, activated (but not resting) macrophages possess a functionally active receptor for folate (FR-ß), but it is not completely clear which subtype of this activated macrophages expresses this receptor and whether the expression of FR-ß is restricted to only one of the macrophage subsets. Although future research needs to be done to investigate FR-ß expression and function within inflamed tissues, the expression of functional FR-ß on tissue macrophages likely occurs during activation. Therefore, expression of FR-ß on activated macrophages holds a promising potential for early diagnosis and better analysis of optimal treatment regiments of vascular diseases in association with systemic diseases.