Annexin V expression on CD4+ T cells with regulatory function.

Regulatory T (Treg) cells induce immunologic tolerance by suppressing effector functions of conventional lymphocytes in the periphery. On the other hand, immune silencing is mediated by recognition of phosphatidylserine (PS) on apoptotic cells by phagocytes. Here we describe expression of the PS-binding protein Annexin V (ANXA5) in CD4+  CD25hi Treg cells at the mRNA and protein levels. CD4+  ANXA5+ T cells constitute about 0·1%-0·6% of peripheral blood CD3+ T cells, exhibit co-expression of several Treg markers, such as Forkhead box P3, programmed cell death protein-1, cytotoxic T-lymphocyte antigen-4 and CD38. In vitro, ANXA5+ Treg cells showed enhanced adhesion to PS+ endothelial cells. Stimulated by anti-CD3 and PS+ syngeneic antigen-presenting cells CD4+  ANXA5+ T cells expanded in the absence of exogenous interleukin-2. CD4+  ANXA5+ T cells suppressed CD4+  ANXA5- T-cell proliferation and mammalian target of rapamycin phosphorylation, partially dependent on cell contact. CD4+  ANXA5+ T-cell-mediated suppression was allo-specific and accompanied by an increased production of anti-inflammatory mediators. In vivo, using a model of delayed type hypersensitivity, murine CD4+  ANXA5+ T cells inhibited T helper type 1 responses. In conclusion, we report for the first time expression of ANXA5 on a subset of Treg cells that might bridge classical regulatory Treg function with immune silencing.

Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions.

The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8(+) T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8(+) T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-gamma, and macrophage inflammatory protein-1alpha by CD8(+) T cells. MCs regulated antigen-specific CD8(+) T cell cytotoxicity by increasing granzyme B expression and by promoting CD8(+) T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8(+) T cell proliferation, and MCs regulated CD8(+) T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8(+) T cells.

Transcription regulates HIF-1α expression in CD4(+) T cells.

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates the metabolic adaptation of cells to hypoxia and T-helper cell fate. However, HIF-1α regulation in CD4(+) T cells (T cells) remains elusive. Here we observed that depletion of oxygen (O2⩽2%) alone was not sufficient to induce HIF-1α expression in T cells. However, when hypoxic T cells were stimulated, HIF-1α was expressed and this was dependent on nuclear factor-κB- and nuclear factor of activated T cell (NFAT)-mediated transcriptional upregulation of Hif-1α mRNA. HIF-1α upregulation could be blocked by drugs inhibiting NF-κB, NFAT or mammalian target of rapamycin precluding CD4(+) T-cell stimulation or translation in T cells, as well as by blocking transcription. CD3, CD28, phorbol-12-myristat-13-acetat (PMA) or ionomycin-stimulated T cells did not express HIF-1α under normoxic conditions. In conclusion, regulation of HIF-1α expression in CD4(+) T cells in hypoxia gravely relies on its transcriptional upregulation and subsequent enhanced protein stabilization.

I787 provides signals for c-Kit receptor internalization and functionality that control mast cell survival and development.

Mast cell (MC) differentiation, survival, and activation are controlled by the membrane tyrosine kinase c-Kit upon interaction with stem cell factor (SCF). Here we describe a single point mutation induced by N-ethyl-N-nitrosurea (ENU) mutagenesis in C57BL/6J mice-an A to T transversion at position 2388 (exon 17) of the c-Kit gene, resulting in the isoleucine 787 substitution by phenylalanine (787F), and analyze the consequences of this mutation for ligand binding, signaling, and MC development. The Kit(787F/787F) mice carrying the single amino acid exchange of c-Kit lacks both mucosal and connective tissue-type MCs. In bone marrow-derived mast cells (BMMCs), the 787F mutation does not affect SCF binding and c-Kit receptor shedding, but strongly impairs SCF-induced cytokine production, degranulation enhancement, and apoptosis rescue. Interestingly, c-Kit downstream signaling in 787F BMMCs is normally initiated (Erk1/2 and p38 activation as well as c-Kit autophosphorylation) but fails to be sustained thereafter. In addition, 787F c-Kit does not efficiently mediate Cbl activation, leading to the absence of subsequent receptor ubiquitination and impaired c-Kit internalization. Thus, I787 provides nonredundant signals for c-Kit internalization and functionality.

The influence of regulatory T cells and diurnal hormone rhythms on T helper cell activity.

Symptoms of diseases such as rheumatoid arthritis, which is T helper 1 (Th1) dependent, and asthma, which is T helper 2 (Th2) dependent, are influenced by diurnal rhythms and natural regulatory T cells (nTreg). However, the mechanisms responsible for the diurnal rhythm of disease activity have not been identified and it is unclear whether nTreg activity is diurnal rhythm-dependent. We therefore investigated whether a 24-hr diurnal cycle affected the ability of various helper T-cell populations to generate immunomodulatory and pro-inflammatory cytokines, as well as its suppression by nTreg cells. Using a within-subject crossover design, sleep versus continuous wakefulness was compared over a 24-hr period in healthy young volunteers under defined environmental conditions. Venous blood was drawn periodically every 5 hr and the function of T cells was explored in vitro. We demonstrated that interleukin (IL)-2, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and IL-10 secretion by naïve CD4(+) T cells follows a diurnal rhythm. Furthermore, multiple regression analysis, as well as subsequent in vitro experiments, suggested that serum levels of cortisol and prolactin are part of the underlying mechanism. Additionally, we observed that nTreg suppressed the secretion of IFN-γ, IL-2 and TNF-α, but not the secretion of IL-4, IL-6, IL-10 and IL-17A. However, the abrogation of IL-2 release was reversed upon inhibiting CD25 on nTreg. Highly purified nTreg secreted IL-6, IL-10 and IL-17A, but not IL-2, IL-4, IFN-γ or TNF-α. Taken together, our results demonstrate that hormones and nTreg modulate the diurnal rhythm of T helper cell activity.

Sleep, immunity, and circadian clocks: a mechanistic model.

The lack of sufficient amounts of sleep is a hallmark of modern living, and it is commonly perceived that in the long run this makes us sick. An increasing amount of scientific data indicate that sleep deprivation has detrimental effects on immune function. Conversely, immune responses feedback on sleep phase and architecture. Several studies have investigated the impact of short-term sleep deprivation on different immune parameters, whereas only a few studies have addressed the influence of sleep restriction on the immune system. In many cases, sleep deprivation and restriction impair immune responses by disrupting circadian rhythms at the level of immune cells, which might be a consequence of disrupted endocrine and physiological circadian rhythms. Little is known about the mechanisms underlying the circadian regulation of immunity, but recent studies have suggested that local as well as central circadian clocks drive the rhythms of immune function. In this review, we present a mechanistic model which proposes that sleep (through soluble factors and body temperature) primes immune cells on the one hand, and, on the other hand, provides a timing signal for hematopoietic circadian clocks. We hypothesize that chronic sleep disruption desynchronizes these clocks and, through this mechanism, deregulates immune responses.

HIF-1α- and hypoxia-dependent immune responses in human CD4+CD25high T cells and T helper 17 cells.

The central oxygen sensitive transcription factor HIF-1α has been implicated in the differentiation of n(T(reg)) and Th17 cells and to orchestrate metabolic changes of activated T cells. However, data on the functional relevance of HIF-1α and Hox, in general, for nT(reg)-suppressive activity and T cell function in primary human cells are still missing. Therefore, we analyzed the effect of Hox and HIF-1α on human T(res), n(Treg), and Th17 cells. Under Hox, nT(reg)-mediated suppression of T(res) proliferation, CD25 expression, and secretion of IFN-γ were significantly reduced, whereas expression levels of VEGF, TNF-α, and IL-10 were significantly increased. In contrast to observations in mice, Th17 lineage commitment, as determined by RORγt expression, was not affected by activation or inhibition of HIF-1α expression using DMOG or YC-1 treatment, respectively. Nevertheless, the secretion of IL-17A was increased by DMOG and reduced by YC-1 under Th17-skewing conditions in a dose- dependent manner. In conclusion, Hox and HIF-1α substantially influence human T cell-mediated immune responses by modulation of nT(reg)-suppressive function and IL-17A secretion by Th17 cells.

Ectonucleotidase CD38 demarcates regulatory, memory-like CD8+ T cells with IFN-γ-mediated suppressor activities.

Regulatory CD8(+) T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8(+) T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38(high) T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8(+)CD38(high) (CD44(+)CD122(+)CD62L(high)) lymphocytes suppress CD4(+) effector T-cell proliferation in an antigen-non specific manner via IFN-γ. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-ß and granzyme B release. IL-15 potentiates the suppressive activity of CD8(+)CD38(high) T cells and controls their survival and expansion. In humans CD8(+)CD38(high) T cells inhibit CD4(+) effector T cell proliferation. In vivo, CD8(+)CD38(high), but not CD8(+)CD38(-) T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8(+)CD38(high) T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8(+)CD38(high) T cells as potential inhibitors of excessive immune responses.

Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum.

The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo. TGF-beta induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.

Chlamydia pneumoniae multiply in neutrophil granulocytes and delay their spontaneous apoptosis.

The obligate intracellular bacterial pathogen Chlamydia pneumoniae (Cp) is responsible for a range of human diseases, including acute respiratory infection. Although experimental intratracheal infection with Cp results in a massive recruitment of neutrophil granulocytes (polymorphonuclear neutrophils (PMN)), the role of these cells in the defense against Cp is unclear. In this study the interactions of PMN with Cp were investigated. In vitro coincubation experiments showed that human granulocytes were able to internalize Chlamydia in an opsonin-independent manner. Importantly, phagocytosed Cp were not killed; the ingested bacteria survived and multiplied within PMN. Although uninfected granulocytes became apoptotic within 10 h, infected PMN survived up to 90 h. Coincubation with Cp significantly decreased the ratio of apoptotic PMN, as detected by morphological analysis, annexin V, and TUNEL staining. The observed antiapoptotic effect was associated with a markedly lower level of procaspase-3 processing and, consequently, reduced caspase-3 activity in infected PMN. LPS was found as a major, but not exclusive, component responsible for the observed antiapoptotic effect. Chlamydia LPS affected PMN apoptosis both by acting directly on the cells and by inducing the autocrine production of the antiapoptotic cytokine IL-8. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, Cp can extend the life span of neutrophil granulocytes, making them suitable host cells for survival and multiplication within the first hours/days after infection.