Markus Feuerer

Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters.

Obesity is accompanied by chronic, low-grade inflammation of adipose tissue, which promotes insulin resistance and type-2 diabetes. These findings raise the question of how fat inflammation can escape the powerful armamentarium of cells and molecules normally responsible for guarding against a runaway immune response. CD4+ Foxp3+ T regulatory (Treg) cells with a unique phenotype were highly enriched in the abdominal fat of normal mice, but their numbers were strikingly and specifically reduced at this site in insulin-resistant models of obesity. Loss-of-function and gain-of-function experiments revealed that these Treg cells influenced the inflammatory state of adipose tissue and, thus, insulin resistance. Cytokines differentially synthesized by fat-resident regulatory and conventional T cells directly affected the synthesis of inflammatory mediators and glucose uptake by cultured adipocytes. These observations suggest that harnessing the anti-inflammatory properties of Treg cells to inhibit elements of the metabolic syndrome may have therapeutic potential.

FOXP3 Controls Regulatory T Cell Function through Cooperation with NFAT

Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin αEβ7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. αE −CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, αE-positive subsets (CD25+ and CD25−) displayed an effector/memory phenotype expressing high levels of E/P-selectin–binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, αE-expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue T reg cells

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T (Treg) cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the ‘master regulator’ of adipocyte differentiation, as a crucial molecular orchestrator of VAT Treg cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT Treg cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of Treg cells with unique functions can be precisely targeted to therapeutic ends.

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell–T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell–mediated immunity.

Origin of monocytes and macrophages in a committed progenitor

Monocytes, macrophages and dendritic cells (DCs) are developmentally related regulators of the immune system that share the monocyte-macrophage DC progenitor (MDP) as a common precursor. Unlike differentiation into DCs, the distal pathways for differentiation into monocytes and monocyte-derived macrophages are not fully elucidated. We have now demonstrated the existence of a clonogenic, monocyte- and macrophage-restricted progenitor cell derived from the MDP. This progenitor was a Ly6C+ proliferating cell present in the bone marrow and spleen that generated the major monocyte subsets and macrophages, but not DCs or neutrophils. By in-depth quantitative proteomics, we characterized changes in the proteome during monocyte differentiation, which provided insight into the molecular principles of developing monocytes, such as their functional maturation. Thus, we found that monocytes and macrophages were renewed independently of DCs from a committed progenitor.

How punctual ablation of regulatory T cells unleashes an autoimmune lesion within the pancreatic islets.

CD4(+)Foxp3(+) regulatory T cells (Treg cells) are known to control the progression of autoimmune diabetes, but when, where, and how they exert their influence in this context are questions still under vigorous debate. Exploiting a transgene encoding the human diphtheria toxin receptor, we punctually and specifically ablated Foxp3(+) cells in the BCD2.5/NOD mouse model of autoimmune diabetes. Strikingly, overt disease developed within 3 days. The earliest detectable event was the activation of natural killer (NK) cells directly within the insulitic lesion, particularly the induction of Ifng gene expression within 7 hours of Treg cell ablation. Interferon-gamma had a strong impact on the gene-expression program of the local CD4(+) T effector cell population, unleashing it to aggressively attack the islets, which was required for the development of diabetes. Thus, Treg cells regulate pancreatic autoimmunity in situ through control of a central innate immune system player, NK cells.

Genomic definition of multiple ex vivo regulatory T cell subphenotypes

Regulatory T (Treg) cells that express the Foxp3 transcription factor are essential for lymphoid homeostasis and immune tolerance to self. Other nonimmunological functions of Treg cells, such as controlling metabolic function in adipose tissue, are also emerging. Treg cells originate primarily in the thymus, but can also be elicited from conventional T cells by in vivo exposure to low-dose antigen or homeostatic expansion or by activation in the presence of TGFβ in vitro. Treg cells are characterized by a distinct transcriptional signature controlled in part, but not solely, by Foxp3. For a better perspective on transcriptional control in Treg cells, we compared gene expression profiles of a broad panel of Treg cells from various origins or anatomical locations. Treg cells generated by different means form different subphenotypes and were identifiable by particular combinations of transcripts, none of which fully encompassed the entire Treg signature. Molecules involved in Treg cell effector function, chemokine receptors, and the transcription factors that control them were differentially represented in these subphenotypes. Treg cells from the gut proved dissimilar to cells elicited by exposure to TGFβ in vitro, but instead they resembled a CD103+Klrg1+ subphenotype preferentially generated in response to lymphopenia.

The defect in T-cell regulation in NOD mice is an effect on the T-cell effectors

FoxP3+ regulatory T cells (Tregs) protect against autoimmunity, type 1 diabetes (T1D) in particular, prompting the hypothesis that a deficiency in Tregs is a critical determinant of diabetes susceptibility in NOD mice. However, tests of this hypothesis have yielded contradictory results. We confirmed that NOD mice, compared with reference strains, do not have a primary deficit in Treg numbers in the lymphoid organs, whether in prediabetic mice of any age or in animals with recent-onset diabetes. NOD Tregs did show a defect in standard in vitro T cell suppression assays, particularly at low suppressor/effector ratios. Gene expression profiling revealed the vast majority of transcripts constituting the “Treg signature” to be normally distributed in NOD Tregs versus CD4+ T conventional (Tconv) cells, although there were a few differences affecting one or the other population. According to results from criss-cross experiments, the functional inefficacy was not rooted in NOD Tregs, which suppressed as well as their C57BL/6 (B6) counterparts, but rather in NOD Tconv, which were less prone to suppression than were B6 Tconv cells. They also responded more effectively to anti-CD3/28 monoclonal antibody (mAb) stimulation in vitro or to a natural pancreatic antigen in vivo. This difference was independent of autoimmune inflammation, did not map to the idd3 region, and was not due to the overproduction of interleukin-21 in NOD mice. That the immune dysregulation in this T1D model is rooted in the ability of effector T cells to be regulated, rather than in Tregs themselves, has implications for proposed therapeutic interventions.

Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients

Previous studies with animal tumors showed that bone marrow (BM) is a privileged site where potentially lethal tumor cells are controlled in a dormant state by the immune system. Here, we investigated BM of breast cancer patients with respect to tumor cell content, immune activation status and memory T‐cell content. BM‐derived cells from primary operated breast cancer patients (n = 90) were compared with those from healthy donors (n = 10) and also with cells from respective blood samples. Cytokeratin 19‐positive tumor cells were detected by nested polymerase chain reaction. Three‐color flow cytometry was used to identify numbers and activation state of T cells, natural killer (NK) cells, monocytes/macrophages and subsets by a panel of monoclonal antibodies (mAbs). The proportion of memory T cells among the CD4 and CD8 T cells was much higher in BM of cancer patients than in healthy donors (p < 0.001). The extent of memory T‐cell increase was related to the size of the primary tumor. Patient‐derived BM memory CD8 T cells could be shown to contain specific HLA‐A2/Her‐2/neu369–377 tetramer binding cells. Patients with disseminated tumor cells in their BM had more memory CD4 T cells and more CD56+ CD8+ cells than patients with tumor cell‐negative BM. Only some of the immunological changes seen in BM samples of cancer patients were also detectable in peripheral blood samples. Our hypothesis that BM is a special compartment for immunological memory and tumor dormancy is supported by the above findings. The overall results reveal that BM is a valuable additional compartment for immune diagnosis in pathological conditions and possibly for follow‐up treatment strategies.