Multidimensional analyses of proinsulin peptide-specific regulatory T cells induced by tolerogenic dendritic cells

Induction of antigen-specific regulatory T cells (Tregs) in vivo is the holy grail of current immune-regulating therapies in autoimmune diseases, such as type 1 diabetes. Tolerogenic dendritic cells (tolDCs) generated from monocytes by a combined treatment with vitamin D and dexamethasone (marked by CD52hi and CD86lo expression) induce antigen-specific Tregs. We evaluated the phenotypes of these Tregs using high-dimensional mass cytometry to identify a surface-based T cell signature of tolerogenic modulation. Naïve CD4+ T cells were stimulated with tolDCs or mature inflammatory DCs pulsed with proinsulin peptide, after which the suppressive capacity, cytokine production and phenotype of stimulated T cells were analysed. TolDCs induced suppressive T cell lines that were dominated by a naïve phenotype (CD45RA+CCR7+). These naïve T cells, however, did not show suppressive capacity, but were arrested in their naïve status. T cell cultures stimulated by tolDC further contained memory-like (CD45RA-CCR7-) T cells expressing regulatory markers Lag-3, CD161 and ICOS. T cells expressing CD25lo or CD25hi were most prominent and suppressed CD4+ proliferation, while CD25hi Tregs also effectively supressed effector CD8+ T cells.We conclude that tolDCs induce antigen-specific Tregs with various phenotypes. This extends our earlier findings pointing to a functionally diverse pool of antigen-induced and specific Tregs and provides the basis for immune-monitoring in clinical trials with tolDC.Nephrolog

High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity

Objective A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.Design Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.Results We discovered that a previously unappreciated innate lymphocyte population (Lin(-)CD7+(C)D127(-)CD56(+)CD45RO(+)) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103(+)CD69(+)) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and gamma delta T cells with highly similar activated (HLA-DR(+)CD38(+)PD(-)1(+)) phenotypes. Remarkably, activated gamma delta T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and gamma delta T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.Conclusion This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.Surgical oncolog

The contribution of cytomegalovirus infection to immune senescence is set by the infectious Dose

Stemcel biology/Regenerative medicine (incl. bloodtransfusion

Systems analysis and controlled malaria infection in Europeans and Africans elucidate naturally acquired immunity

Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161(+)CD4(+) T cells and interferon-gamma production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4(+) T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.Malaria immunity can be acquired through natural infection, but the correlates of protection are still being determined. Yazdanbakhsh and colleagues combine experimental infection of volunteers with Plasmodium falciparum with systems analysis to throw light on the nature of protective immune responses.Radiolog

Cholecystokinin octapeptide activates an opioid mechanism in the guinea-pig ileum: A possible role for substance P

To test the hypothesis that excitatory peptides release endogenous opioids from the myenteric plexus longitudinal muscle (MPLM) preparation of the guinea-pig ileum (GPI), the effect of cholecystokinin octapeptide (CCK8) was studied in the absence and presence of the opioid antagonist naloxone. The maximum height of the contracture induced by CCK8 was not altered by the presence of naloxone in the incubation medium, however, the subsequent sustained excitation was clearly increased. This effect is interpreted as being a result of the release of endogenous opioids during the first moments of the CCK8-evoked excitation of the plexus. CCK8 still induced neurogenic contractures in the presence of atropine; these contractures were probably mediated by the release of substance P. Naloxone was used to evidence the opioid control of the CCK8-induced release of substance P. Desensitization to the effect of substance P reduced the action of CCK8 and also abolished the non-cholinergic contractures evoked by CCK8 and the subsequent effect of naloxone. These facts suggest the release of endogenous opioids within the plexus in response to the neurally mediated excitatory action of CCK8.Peer Reviewe

Neuropeptide Y is an inhibitor of neural function in the myenteric plexus of the guinea-pig ileum

Peer Reviewe

Neural activation of opioid mechanisms in guinea pig ileum by excitatory peptides

The opioid antagonist naloxone added after contractions induced by excitatory peptides on the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum evoked a contracture with intensity dependent on the concentration of the antagonist and on the interval of time that elapsed after the cessation of the excitation induced by the peptides. A direct relationship was found between the component of the peptide-induced contraction that appeared to be neurally mediated and the magnitude of the subsequent contracture induced by naloxone. Moreover, the opioid agonist FK 33-824 decreased the contractile effect of the excitatory peptides and also increased the height of the naloxone-induced contractures. That effect was particularly evident when the higher doses of the peptides were studied. In the presence of atropine naloxone elicited small contractures when added after peptides with primarily neural actions, including corticotropin-releasing factor, neurotensin and cholecystokinin octapeptide 26-33 sulfated form. The naloxone-induced contracture appears to be mediated mainly by the release of acetylcholine, and to a minor extent by another spasmogenic substance from the myenteric plexus. It is concluded that endogenous opioids modulate, i.e., inhibit the release of those excitatory endogenous transmitters triggered by the effect of excitatory peptides upon the myenteric plexus.Peer Reviewe

Opiate activity of peptides derived from the three opioid peptide families on the rat vas deferens

The inhibitory activity of opioid peptides derived from pro-opiomelano-cortin (POMC), pro-enkephalin A and pro-enkephalin B (= pro-dynorphin) on the electrically evoked twitch of the rat vas deferens (RVD) was evaluated. The POMC-derived β-endorphin exhibits the greatest potency on this preparation. In addition, all peptides derived from pro-enkephalin A show full agonistic activity with BAM-22P and peptide E as the most potent peptides. In contrast, the majority of peptides derived from pro-enkephalin B (= pro-dynorphin) were essentially inactive on this tissue. Moreover, no antagonistic properties of these peptides were demonstrable in this preparation; thus the opioid receptors present in the RVD (putative epsilon receptors) might not possess any particular affinity for the pro-enkephalin B derived peptides.Peer Reviewe

Analysis of promoter regions regulating basal and interleukin-4-inducible expression of the human CB1 receptor gene in T lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) 3086 to 2490 and nts 1950 to 1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts 648 and 559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt 2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R()-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)- 1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N- 4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells