Stochastics of Cellular Differentiation Explained by Epigenetics: The Case of T-Cell Differentiation and Functional Plasticity.

Epigenetic marks including histone modifications and DNA methylation are associated with the regulation of gene expression and activity. In addition, an increasing number of non-coding RNAs with regulatory activity on gene expression have been identified. Alongside, technological advancements allow for the analysis of these mechanisms with high resolution up to the single-cell level. For instance, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) simultaneously probes for chromatin accessibility and nucleosome positioning. Thus, it provides information on two levels of epigenetic regulation. Development and differentiation of T cells into functional subset cells including memory T cells are dynamic processes driven by environmental signals. Here, we briefly review the current knowledge of how epigenetic regulation contributes to subset specification, differentiation and memory development in T cells. Specifically, we focus on epigenetic mechanisms differentially active in the two distinct T cell populations expressing αß or γδ T cell receptors. We also discuss examples of epigenetic alterations of T cells in autoimmune diseases. DNA methylation and histone acetylation are subject to modification by several classes of 'epigenetic modifiers', some of which are in clinical use or in preclinical development. Therefore, we address the impact of some epigenetic modifiers on T-cell activation and differentiation, and discuss possible synergies with T cell-based immunotherapeutic strategies.

CD20-Specific Immunoligands Engaging NKG2D Enhance γδ T Cell-Mediated Lysis of Lymphoma Cells.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

Effector memory T­cells in the pathogenesis of ANCA-associated vasculitides.

Patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) have an expansion of effector memory T­cells in peripheral blood. The enlarged effector memory cell population contains distinct cell subsets, including T­helper type 1 (Th1) CD4+ T­cells lacking co-stimulatory CD28 expression and Th17 cells in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and Th2 type and Th17 cells in eosinophilic granulomatosis with polyangiitis (EGPA). The cytokine response of autoreactive proteinase 3 (PR3)-specific effector memory T­cells is skewed towards an increase of Th2 type, Th17, and Th22 cell fractions in GPA. Anomalous effector memory CD4+ T­cell co-stimulation is suggested by the aberrant expression of P­selectin glycoprotein ligand-1, ß­2 integrin, chemokine receptors, natural-killer group 2 member D (NKG2D) and other activating receptors. The increased expression of these receptors is accompanied by T­cell activation and migration to inflamed tissues. T­cells are abundant and secrete proinflammatory cytokines in inflammatory lesions in AAV. The T­cell mediated tissue damage correlates with renal outcome, whereas B­cell infiltration does not. Activation of lesional CD4+NKG2D+ effector memory T­cells is independent of the antigen; moreover, CD4+NKG2D+ effector memory T­cells display NK-cell-like cytotoxicity towards microvascular endothelial cells in vitro. Thus, effector memory T­cells play an important role in tissue damage and disease progression in AAV. Sequentially administered or combined with B­cell depleting therapy, T­cell-directed therapies, especially those directed against effector memory CD4+ T­cells, may further improve the outcome and help to achieve long-term remissions in AAV.

[Effector memory T­cells in the pathogenesis of ANCA-associated vasculitides. German version]. / Effektor-Memory-T­Zellen in der Pathogenese von ANCA-assoziierten Vaskulitiden.

Patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) have an expansion of effector memory T­cells in peripheral blood. The enlarged effector memory cell population contains distinct cell subsets, including T­helper type 1 (Th1) CD4+ T­cells lacking co-stimulatory CD28 expression and Th17 cells in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and Th2 type and Th17 cells in eosinophilic granulomatosis with polyangiitis (EGPA). The cytokine response of autoreactive proteinase 3 (PR3)-specific effector memory T­cells is skewed towards an increase of Th2 type, Th17 and Th22 cell fractions in GPA. Anomalous effector memory CD4+ T­cell co-stimulation is suggested by the aberrant expression of P­selectin glycoprotein ligand­1, beta­2 integrin, chemokine receptors, natural-killer group 2 member D (NKG2D) and other activating receptors. The increased expression of these receptors is accompanied by T­cell activation and migration to inflamed tissues. The T­cells are abundant and secrete proinflammatory cytokines in inflammatory lesions in AAV. The T­cell mediated tissue damage correlates with renal outcome, whereas B-cell infiltration does not. Activation of lesional CD4+NKG2D+ effector memory T­cells is independent of the antigen; moreover, CD4+NKG2D+ effector memory T­cells display NK-cell-like cytotoxicity towards microvascular endothelial cells in vitro. Thus, effector memory T­cells play an important role in tissue damage and disease progression in AAV. Sequentially administered or combined with B-cell depleting therapy, T­cell-directed therapies, especially those directed against effector memory CD4+ T­cells, may further improve the outcome and help to achieve long-term remission in AAV.

Lysosome-Related Effector Vesicles in T Lymphocytes and NK Cells.

Lysosome-related secretory organelles combine metabolic functions of conventional lysosomes with an inducible secretory potential. Specialized variants of such bi-functional organelles are present in several haematopoietic cell types that store, mobilize and/or secrete effector proteins, for example in mast cells, macrophages or cytotoxic effector cells. In the case of T lymphocytes and NK cells, it was believed that secretory lysosomes serve as a common storage and transport compartment for the most relevant cytotoxic effector proteins including FasL, perforin, granzymes and granulysin. However, recent observations suggest that cytotoxic effector cells might be able to mobilize two distinct lysosomal entities in order to react to differential stimulation with either FasL surface appearance or degranulation-associated release of perforin and granzymes. This assumption is supported by the proteomic characterization of enriched organelles from T and NK cells. FasL-associated light lysosomes biochemically segregate from morphologically distinct heavy lysosomes that preferentially contain granzymes, perforin and mature granulysin. Here, we briefly summarize the current knowledge about cargo proteins that are stored and transported in secretory vesicles and how these vesicles might be generated and mobilized. In addition, we describe common features and major differences of the two distinct effector organelles and discuss how these observations might expand existing models of cytotoxic effector function.

Inhibition of human γδ T cell proliferation and effector functions by neutrophil serine proteases.

Human peripheral blood γδ T cells expressing the Vγ9Vδ2 T cell receptor are activated by microbial or endogenous pyrophosphate antigens and indirectly by nitrogen-containing bisphosphonates. Apart from proliferation, such phosphoantigens induce proinflammatory cytokine production including TNF-α and IFN-γ and trigger cytotoxic effector function. Neutrophil granulocytes are known to modulate T cell activation. The neutrophil serine proteases proteinase 3, elastase and cathepsin G have multiple potential targets and promote microbial killing. In this study, we investigated the effect of the three serine proteases on the in vitro proliferation and effector functions of γδ T cells cultured in serum-free medium. All three proteases inhibited the proliferative activity, suppressed the cytokine production and decreased the cytotoxicity of γδ T cells. Further studies indicated that proteolytic cleavage of IL-2 and modulation of butyrophilin 3A1 (CD277) expression might contribute to the overall inhibition.

In vitro comparison of different carrier materials with rat bone marrow MSCs.

OBJECTIVES: Injectable or implantable scaffolds seeded with autologous chondrogenic cells may represent a promising option for treatment of cartilage defects in the future. Current problems with the autologous chondrocyte implantation including dedifferentiation and the development of fibrocartilage suggest the use of alternative chondrogenic cell sources such as mesenchymal stromal cells (MSCs). The aim of this study was to compare the early effects of different scaffolds on the proliferation and metabolic activity of chondrogenic MSCs in vitro. MATERIALS AND METHODS: Multipotent stromal cells were isolated from rat bone marrow, phenotyped by flow cytometry, and differentiated into distinct lineages proved by lineage-specific staining and gene expression (RT-PCR) pattern. Cell proliferation on Tutodent® Membrane, Bio-Gide®, TissuFleece E, and Belotero® Soft was quantified by the MTT and WST-1 assay and direct determination of total cell numbers. Potential cytotoxic effects of eluates obtained from the materials were quantified by lactate dehydrogenase (LDH) and 5-bromo-2-deoxyuridine (BrdU) assay. RESULTS: TissuFleece E displayed the best results regarding cell proliferation on the biomaterials and metabolic activity (MTT, WST-1) (p < 0.001). Yet, the eluates of TissuFleece E caused an increased LDH release and lower values in the BrdU test. Cell proliferations on Bio-Gide®, Tutodent® Membrane, and Belotero® Soft were similar to the control. The eluates of Belotero® Soft exhibited the highest LDH release and lowest values in the BrdU assay (p < 0.05). CONCLUSIONS: Our results support the use of Tissufleece E as scaffold for chondrogenic rat MSCs. However, it should be prewashed with culture medium before seeding of the cells. CLINICAL RELEVANCE: Tissufleece E may serve as a promising carrier material for chondrogenic MSCs for cartilage tissue engineering attempts.

Generation of soluble NKG2D ligands: proteolytic cleavage, exosome secretion and functional implications.

The activating natural killer group 2 member D (NKG2D) receptor is expressed on NK cells, cytotoxic T cells and additional T cell subsets. Ligands for human NKG2D comprise two groups of MHC class I-related molecules, the MHC class I chain-related proteins A and B (MICA/B) and 6 UL16-binding proteins (ULBP1-6). While NKG2D ligands are absent from most normal cells, expression is induced upon stress and malignant transformation. In fact, most solid tumours and leukaemia/lymphomas constitutively express at least one NKG2D ligand and thereby are susceptible to NKG2D-dependent immunosurveillance. However, soluble NKG2D ligands are released from tumour cells and can down-modulate NKG2D activation as a means of tumour immune escape. In some tumour entities, levels of soluble NKG2D ligands in the serum correlate with tumour progression. NKG2D ligands can be proteolytically shed from the cell surface or liberated from the membrane by phospholipase C in the case of glycosylphosphatidylinositol (GPI)-anchored molecules. Moreover, NKG2D ligands can be secreted in exosomal microvesicles together with other tumour-derived molecules. Depending on the specific tumour/immune cell setting, these various forms of soluble and/or exosome-bound NKG2D ligands can exert multiple effects on NKG2D/NKG2D ligand interactions. In this review, we focus on the role of various proteases in the shedding of human NKG2D ligands from tumour cells and discuss the not completely unanimous reported functional implications of soluble and exosome-secreted NKG2D ligands for immunosurveillance.

Markers of operational immune tolerance after pediatric liver transplantation in patients under immunosuppression.

A prospective identification of the estimated 20-50% of pediatric LTX recipients developing operational tolerance would be of great clinical advantage. So far markers of immune tolerance - T-cell subpopulations or gene expression profiles - have been investigated only retrospectively in successfully weaned patients. Fifty children aged 8-265 months (median 89) were investigated 1-180 months (median 44) after LTX under ongoing immunosuppression. T-cell subpopulations were measured during regular post-transplant visits using FACS (Vδ1- vs. Vδ2-γδ-T cells and Tregs). A Vδ1/Vδ2-γδ-T-cell ratio ≥1.42 previously reported in operational tolerance was found in 12 of 50 (24%) patients. In analogy, a Treg count ≥44 per µL was found in 35 of 50 (70%) patients and a Treg proportion ≥2.23% of CD3(+) -T cells in 39 of 50 (78%) patients. Only 9 of 50 patients (18%) fulfilled both criteria. The parameters Vδ1/Vδ2-γδ-T-cell ratio and Tregs were not significantly correlated to each other or with donor type or immunosuppression. Vδ1/Vδ2-γδ-T-cell ratio was more stable in serial examinations compared with Treg analyses. The observed proportion of 18% pediatric LTX patients with potential operational tolerance is in accordance with previous reports. However, clinical experience shows that rejections may happen even after long-time weaning of immunosuppression. This suggests that operational tolerance is a dynamic process, with uncertain prediction by Vδ1/Vδ2-γδ-T-cell ratio and/or Tregs under immunosuppression.

The multifunctionality of human Vγ9Vδ2 γδ T cells: clonal plasticity or distinct subsets?

The dominant subset of γδ T cells in human peripheral blood expresses Vγ9 paired with Vδ2 as variable TCR elements. Vγ9Vδ2 T cells recognize pyrophosphates derived from the microbial non-mevalonate isoprenoid biosynthesis pathway at pico- to nanomolar concentrations. Structurally related pyrophosphates are generated in eukaryotic cells through the mevalonate pathway involved in protein prenylation and cholesterol synthesis. However, micromolar concentrations of endogenous pyrophosphates are required to be recognized by Vγ9Vδ2 T cells. Such concentrations are not produced by normal cells but can accumulate upon cellular stress and transformation. Therefore, many tumour cells are susceptible to γδ T cell-mediated lysis owing to the overproduction of endogenous pyrophosphates. This explains why Vγ9Vδ2 T cells contribute to both anti-infective and anti-tumour immunity. Ex vivo analysed Vγ9Vδ2 T cells can be subdivided on the basis of additional surface markers, including chemokine receptors and markers for naïve and memory T cells. At the functional level, Vγ9Vδ2 T cells produce a broad range of cytokines, display potent cytotoxic activity, regulate αß T cell responses, and - quite surprisingly - can act as professional antigen-presenting cells. Thus, an exceptional range of effector functions has been assigned to a population of T cells, which all recognize invariant exogenous or endogenous pyrophosphates that are not seen by any other immune cell. Here, we discuss whether this plethora of effector functions reflects the plasticity of individual Vγ9Vδ2 T cells or can be assigned to distinct subsets.

Aminobisphosphonates and Toll-like receptor ligands: recruiting Vγ9Vδ2 T cells for the treatment of hematologic malignancy.

Gamma delta (γδ) T cells are intrinsically important for preventing the development and progression of hematologic cancers. These innate T cells are particularly suited for the application of cancer therapy due to the fact they: 1) recognize transformed cells independent of antigen processing or presentation by classical MHC molecules, and 2) embody the anti-tumour effector functions of both NK cells and cytotoxic T cells. It was serendipitously discovered that aminobisphosphonates (ABP), a class of drugs used as adjuvant cancer therapy for the treatment of malignant osteolytic bone disease, have the unexpected side-effect of potently activating the antitumour effector functions of human peripheral γδ T cells. Such beneficial therapeutic synergisms are rare, and no time has been wasted to determine how to best harness the anti-cancer potential of γδ T cells and ABP. Despite promising experimental results, the full clinical potential of this immunotherapeutic strategy has been hampered by the subversive strategies employed by cancer cells to obstruct activation of anti-tumour immune responses. These include the promotion of regulatory T cells (Tregs) that maintain tumour tolerance and the corruption of dendritic cell (DC) function and maturation. Toll-like receptor (TLR) agonists have a long history of breaking free of tumour-induced immune-suppression by resetting DC function and abrogating Treg induced tolerance. This review presents data to support the notion that TLR signalling may perfectly complement the anti-tumour synergy of ABP and activated γδ T cells, and this combined innate artillery could provide the necessary ammunition to topple malignancy's stronghold on the immune system.

[T-cells in ANCA-associated vasculitis]. / T-Zellen bei ANCA-assoziierten Vaskulitiden.

CD4+NKG2D+effector memory T-cell-mediated endothelial cell damage and a Th17 dominated cytokine profile of PR3-specific T-cells may play an important role in the pathogenesis of granulomatosis with polyangiitis (Wegener's). The anti-IL-5 antibody mepolizumab (anti-IL-5) induces remission in Churg-Strauss syndrome.

Functional expression of NOD2 in freshly isolated human peripheral blood γδ T cells.

γδ T cells play an important role in anti-infective immunity. The major subset of human γδ T cells selectively recognizes phosphorylated bacterial metabolites of the isoprenoid biosynthesis pathway, so-called phosphoantigens. The activation of γδ T cells is modulated by functionally expressed innate immune receptors, notably Toll-like receptor 2 and 3. It was also reported that in vitro expanded γδ T cells respond to muramyl dipeptide (MDP), the minimal peptidoglycan motif activating the nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, although it is unknown whether ex vivo isolated human γδ T cells express functional NOD2. Here, we report that freshly isolated, highly purified peripheral blood γδ T cells express NOD2 mRNA and detectable amounts of NOD2 protein. The biologically active MDP L-D isomer but not the inactive D-D isomer augmented the interferon-γ (IFN-γ) secretion in phosphoantigen-stimulated peripheral blood mononuclear cells. Moreover, a moderate but reproducible and statistically significant increase in IFN-γ secretion was also observed when highly purified peripheral blood γδ T cells were activated by T cell receptor cross-linking in the presence of MDP. Taken together, our results indicate that in addition to the T cell receptor and Toll-like receptors, circulating human γδ T cells express NOD2 as a third class of pattern recognition receptor for sensing bacterial products.

Modulation of CD4+ T-cell activation by CD95 co-stimulation.

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

Human NK cells require caspases for activation-induced proliferation and cytokine release but not for cytotoxicity.

Natural killer (NK) cells are innate immune cells involved in antiviral defence and tumour surveillance. To fulfil these tasks, NK cells make use of two major effector functions, cytokine and chemokine release and cytotoxicity. In addition, NK cells proliferate in response to cytokines such as IL-2. NK cells possess a large array of activating and inhibitory receptors and their activation demands a complex crosstalk between those receptors. The signalling pathways leading to NK-cell activation are a field of intensive research. The first clue for signal specificity was provided by studies showing that a pathway leading to NF-κB activation selectively induces cytokine release, but is dispensable for cytotoxicity. Here, we demonstrate that in human NK cells caspase activity is required for the upregulation of select activation markers and IFN-γ and TNF production, but not for cytotoxicity. Interestingly, caspases have previously been linked in T cells to the same mechanism of NF-κB induction that is active in NK cells. Moreover, we provide evidence that caspases are involved in IL-2-induced proliferation. Thus, our data provide the basis for a novel approach using caspase inhibitors to generate cytotoxic NK cells, while simultaneously suppressing cytokine release.

Human gammadelta T cells produce the protease inhibitor and antimicrobial peptide elafin.

Human gammadelta T cells rapidly secrete pro-inflammatory cytokines in response to T cell receptor-dependent recognition of pyrophosphates produced by many bacteria and parasites. In further support of an important role of gammadelta T cells in the immune defence against infection, human gammadelta T cells have been shown to produce the antimicrobial peptide LL37/cathelicidin. In this study, we have investigated whether gammadelta T cells can produce additional antimicrobial peptides. To this end, we have screened human gammadelta T cell clones by RT-PCR for mRNA expression of a broad range of antimicrobial peptides. While alpha-defensins were absent and beta-defensins (HBD1) present only in rare gammadelta T cell clones, elafin mRNA was induced by supernatant of Pseudomonas aeruginosa grown under static conditions. Elafin is a protease inhibitor that also displays antimicrobial activity. Constitutive intracellular expression of elafin was demonstrated by flow cytometry and Western blot analysis. Furthermore, trappin-2 (pre-elafin) could be immunoprecipitated in cell lysates but also in the supernatant of gammadelta T cells stimulated by Ps. aeruginosa supernatant. Taken together, our studies reveal a novel effector function of gammadelta T cells which might be important for local immune defence.

Toll-like receptor expression and function in subsets of human gammadelta T lymphocytes.

Two subsets of human gammadelta T cells can be identified by T cell receptor (TCR) V gene usage. Vdelta2Vgamma9 T cells dominate in peripheral blood and recognize microbe- or tumour-derived phosphoantigens. Vdelta1 T cells are abundant in mucosal tissue and recognize stress-induced MHC-related molecules. Toll-like receptors (TLRs) are known to co-stimulate interferon-gamma (IFN-gamma) production in peripheral blood gammadelta T cells and in Vdelta2Vgamma9 T cell lines. By microarray analysis, we have identified a range of genes differentially regulated in freshly isolated gammadelta T cells by TCR versus TCR plus TLR3 stimulation. Furthermore, we have investigated TLR expression in freshly isolated Vdelta1 and Vdelta2 subsets and cytokine/chemokine production in response to TLR1/2/6, 3 and 5 ligands. TLR1,2,6,7 RNA was abundantly expressed in both subsets, whereas TLR3 RNA was present at low levels, and TLR5 and 8 RNA only marginally in both subsets. Despite abundant RNA expression, TLR1 was rarely detectable by flow cytometry. In contrast, TLR2 and TLR6 proteins were detected in purified Vdelta1 and Vdelta2 T cells, and TLR3 protein was detected intracellularly in both subsets. TLR1/2/6, 3 and 5 ligands co-stimulated the IFN-gamma and chemokine secretion in TCR-activated Vdelta1 and Vdelta2 subsets, although the levels of IFN-gamma secreted by Vdelta1 T cells were much lower than those produced by Vdelta2 T cells. Our results reveal comparable expression of TLRs and functional responses to TLR ligands in freshly isolated Vdelta1 and Vdelta2 T cells and underscore the intrinsically different capacity for IFN-gamma secretion of Vdelta1 versus Vdelta2 T cells.

[Autoimmunity. Physiological control mechanisms and pathways to autoimmune disease]. / Autoimmunität: Physiologische Kontrollmechanismen und Wege zur Autoimmunerkrankung.

Immunological tolerance towards self antigens is ensured by at least two different mechanisms, i.e. the deletion of potentially autoreactive T-lymphocytes in the thymus ("negative selection") and the active suppression of unwanted (auto)immune responses in the periphery through regulatory T-cells (Treg). With few exceptions, autoimmune diseases result from a multifactorial disturbance of the physiological immune homeostasis. Underlying mechanisms include a genetic predisposition and an aberrant activation of the immune system due to exogenous stimuli such as infectious microorganisms or endogenous stimuli such as disturbed epithelial barrier function. Microbe-derived Toll-like receptor ligands interfere with the control of immune cell activation at several levels including stimulation of autoreactive B-lymphocytes, promotion of autoantigen presentation to T-lymphocytes, and modulation of the suppressive capacity of Treg. In addition, recent evidence indicates that the newly discovered interleukin-17 producing Th(17) T-cells play an important role in promoting inflammatory reactions and tissue destruction in autoimmune diseases.