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The intrinsic immunosuppressive properties of regulatory T (Treg) cells can be harnessed for therapeutic approaches aiming at down-modulating harmful immune reactions. In this context, expanded type 1 Treg cells (Tr1 cells) specific for ovalbumin (ova-Tr1 cells) have been tested for clinical efficacy in the treatment of autoimmune disorders such as refractory Crohn's disease (CD). The clinical use of these therapeutic products warrants exploration of their mechanism of action. Here, we identified a relationship between the CD activity index and the expression of lytic molecules by the ova-Tr1 cells administered in the previously reported First-in-Man study [Crohn's And Treg cells Study 1 (CATS1) study]. Accordingly, ova-Tr1 cells were found to carry granules containing high levels of lytic molecules, including multiple granzymes and granulysin. These cells displayed a T-cell receptor (TCR)-independent cytotoxic activity, which was preferentially directed toward myeloid cell lines and monocyte-derived dendritic cells. Upon contact with myeloid cells, ova-Tr1 cells induced their apoptosis via a perforin-independent and a granulysin/granzyme-dependent mechanism. As compared to CD8+ cytotoxic T cells, ova-Tr1 cells required more time to lyse target cells and displayed a more gradual lytic activity over time. Notably, this activity was sustained over days resulting in the control of myeloid cell populations at a relatively low ratio. Our study reveals that ova-Tr1 cells are endowed with a sustained cytotoxic activity that relies on a unique combination of granulysin and granzymes and that preferentially eliminates myeloid target cells in a TCR-independent manner. [ABSTRACT FROM AUTHOR]

The intestinal epithelium not only acts as the physical structure that separates the intestinal lumen from the body but also actively participates in intestinal barrier functions. In the past decade, significant progress has been made in the development of culture technologies to maintain intestinal epithelial cells (IECs) as various forms of intestinal organoids. As these organoids allow for restoration of the physiological composition of IECs, they represent suitable models to study the mechanisms of development and differentiation or the molecular basis of functions in specific types of IECs, such as goblet cells, Paneth cells, tuft cells and M cells. In addition, intestinal organoids are now widely used as model systems to investigate the dynamic processes occurring at the host–microbe interface and the mutual interactions between IECs and the cells involved in the maintenance of local immune homeostasis. In this review article, I showcase recent work that has utilized intestinal organoids to study various aspects of intestinal epithelial barrier functions. [ABSTRACT FROM AUTHOR]

Gimap5, a member of the GTPase of the immunity-associated protein family (Gimap), regulates T cell survival. A strong indication of this is found in the diabetes-prone BioBreeding rat (BBDP), where a frameshift mutation in Gimap5 results in T-cell lymphopenia. We have investigated the function of human Gimap5 in T cells. We found that reduction of Gimap5 by RNA interference in Jurkat cells did not affect the number of apoptotic cells whereas transient over-expression of Gimap5 resulted in a major increase in the number of apoptotic cells. The same effect of over-expression was found in naive human T cells purified from blood but not in activated human T cells. This suggests that the apoptosis-inducing effect of Gimap5 over-expression is dependent on the activation status of the cells. Since the apoptosis-inducing effect of Gimap5 was contrary to the expected function of Gimap5 based on the phenotype of BBDP rats, we over-expressed rat wt Gimap5 and Gimap5 with the mutation found in BBDP (Gimap5-lyp). Both versions of rat Gimap5 induced apoptosis when expressed in the rat T-cell line C58(NT)D.1.G.OVAR.1, however, Gimap5-lyp greatly exacerbated cell death. Finally, we detected the subcellular localization of Gimap5 to be at the endoplasmic reticulum and by quantitative PCR, we found that endogenous Gimap5 mRNA is up-regulated in activated T cells. [ABSTRACT FROM AUTHOR]

In unimmunized specific pathogen-free mice, there are unique memory-type CD8+ T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (TCMT) judging from their expression profile of CD44, IL-2Rβ, CD62L and CCR7 cell-surface molecules. Among CD44highCD8+ so-called memory CD8+ T cell population, ASGM1+CD44highCD8+ TCMT, but not ASGM1−CD44highCD8+ memory T cells, produced IFN-γ by stimulation with anti-CD3 mAb. The physiological significance of ASGM1+CD8+ TCMT as early source of IFN-γ was also demonstrated in vivo. Namely, intravenous injection of anti-CD3 mAb (2 μg) resulted in early activation of IFN-γ-producing ASGM1+CD8+ TCMT cells as well as NKT and NK cells. Unexpectedly, however, few IFN-γ-producing CD4+ T cells were detected until 4 h after anti-CD3 mAb administration. Thus, ASGM1+CD8+ TCMT were demonstrated to be early IFN-γ producer, which may be crucial for Th1-dependent cellular immunity. Indeed, co-culture of naive CD4+ T cells with ASGM1+CD8+ TCMT but not ASGM1−CD8+ T cells caused a great acceleration of IFN-γ-producing Th1 cells in vitro. Finally, we found that Th1-prone C57BL/6 mice possessed higher percentage (10%) of ASGM1+CD8+ TCMT in CD8+ T cells compared with that (3%) of Th2-prone BALB/c mice. Moreover, ASGM1+CD8+ TCMT derived from C57BL/6 mice produced higher levels of IFN-γ compared with those from BALB/c mice. Thus, ASGM1+CD8+ TCMT, whose differentiation in vivo is genetically controlled, appear to play a critical role in the control of type 1 immunity, which is essential for therapy of tumors and infectious diseases. [ABSTRACT FROM AUTHOR]

The endocytic and phagocytic activities of a population of MHC IIhi CD11c+ dendritic cell (DC)-like cells in synovium-rich tissues (SRTs) of normal rat paws were compared with CD163+ cells (putative macrophages) from the same tissues and pseudo-afferent lymph DCs, peritoneal macrophages and blood monocytes. Fifty percent of CD11c+ cells and 75% of CD163+ cells isolated from SRT internalized fluorescein-conjugated dextran (FITC-DX). Of these endocytic cells, half of those expressing CD11c, but only 30% of those expressing CD163, were surface MHC class II+ (sMHC II+). CD11c+ cells were more endocytic than monocytes or pseudo-afferent lymph DC, but some CD163+ cells (type A synoviocytes) were found to be highly endocytic. CD163+ cells from SRT were more phagocytic (25%) than the general MHC class II+ population (16%). Of phagocytic cells, 40% of CD163+ cells were sMHC IIvariable and they constituted 60% of all MHC class II+ phagocytic cells. Only 18% of phagocytic MHC II+ cells expressed CD11c and the most of these were MHC IIhi. In comparison, 60% of CD163+ peritoneal macrophages were phagocytic, while blood monocytes were poorly phagocytic. Intracellular MHC class II-rich compartments (MIIC) were prominent in sMHC IIhi cells in SRT but rare in CD163+ cells. Most MHC IIhi CD11c+ cells did not have a detectable MIIC. [ABSTRACT FROM AUTHOR]

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL- protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+CD4+ T cells further up-regulate the IL-2R a chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+CD4+ T cells is not a passive phenomenon but instead initiatesin conjunction with signals through the TCRtheir differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo. [ABSTRACT FROM AUTHOR]

TGF-1 is a powerful regulator of various T-cell functions. However, it has been unclear how the T-cell responsiveness towards TGF-1 is affected by its phenotype or signaling intensity. In the present study, we demonstrate that the phenotype and the TCR-signaling intensity of the responding T-cell as well as the presence of anti-CD28 co-stimulation markedly affects how nave human cord blood T-cells respond to TGF-1. In this report we demonstrate that the strength of the stimulatory signal modifies the T-cell response towards TGF-1. Thus, the greatest anti-proliferative effect of TGF-1 was observed during weak stimulatory conditions (low dose of anti-CD3 with no co-stimulatory signal). However, such anti-proliferative effect was reduced during strong stimulatory signal (high dose of anti-CD3 with a CD28-directed co-stimulatory signal). In addition, our results indicate that CD8+ T-cells are generally more responsive towards TGF-1 than CD4+ T-cells. To our surprise, nave T-cells had a skewed Th1/Tc1 cytokine secretion pattern with high amounts of IL-2, IFN? and TNFa, but low amounts of IL-4, IL-5 and IL-10. TGF1 significantly reduced the secretion of IL-2 and IFN?, but such suppression was partially prevented by anti-CD28-induced co-stimulation. In contrast, the inhibitory effect on IL-5 secretion was unaffected by anti-CD28 co-stimulation. Interestingly, TGF-1 induced IL-10 and TNFa secretion. However, the induction of IL-10 secretion was reduced during optimal stimulatory conditions while TGF-1 further induced TNFa secretion. These data demonstrate that the duration, intensity and type of signaling alters the sensitivity of T-cells to powerful immunological modifying agents like TGF-1. [ABSTRACT FROM AUTHOR]

Antigen cross-presentation is critical in infectious and tumor immunity where cytotoxic T lymphocytes are induced by dendritic cells specifically equipped with cellular machineries to present exogenous antigens with major histocompatibility complex (MHC) class I molecules. To examine molecular mechanisms of antigen cross-presentation, we employed as a model system a murine dendritic cell line DC2.4 capable of presenting soluble antigens such as ovalbumin (OVA) with MHC class I. Here, we demonstrate that exogenously added OVA is accumulated in the endoplasmic reticulum (ER) and late endosomes followed by retrograde transport to the cytoplasm through the Sec61 transporter complexes, and that CHIP functions as an E3 ubiquitinligase for OVA degradation by proteasomes. This mechanism is essentially the same as that known as the ER-associated degradation (ERAD) in the quality control of secretary and membrane proteins. [ABSTRACT FROM AUTHOR]

CD226 (DNAM-1) is an adhesion molecule involved in NK and T cell-mediated cytotoxicity against certain tumors. Here, we have identified the human poliovirus receptor-related (PRR) family members CD155 [poliovirus receptor (PVR)] and CD112 (nectin-2/PRR-2) as the ligands for human CD226. Ectopic expression of human CD155 and/or CD112 rendered mouse BW5147 T cells more susceptible to IL-2-activated T and NK cell-mediated cytotoxicity, and killing was specifically inhibited by anti-CD226 mAb, demonstrating functional interactions of CD226 with CD155 and CD112. Although the binding affinities between soluble CD226 and CD155 or CD112 were comparable, the homophilic interaction of cell-surface CD112 may adversely affect CD226 binding to CD112. We also demonstrate that ligation of CD226 and LFA-1 with their respective ligands cooperates in triggering cytotoxicity and cytokine secretion by T and NK cells. [ABSTRACT FROM AUTHOR]

Upon activation, naive CD4 T cells differentiate into different lineages of effector Th subsets. Each subset is characterized by its unique cytokine profile and biological functions. Th17, a newly described Th subset that produces IL-17, IL-17F and IL-22 in preference to other cytokines, has been shown to play an important role in clearing specific pathogens and in inducing autoimmune tissue inflammations. Over the last 2–3 years, significant progress has been made to understand the development and biological functions of Th17 subset. Transforming growth factor β (TGF) together with IL-6 or IL-21 initiates the differentiation while IL-23 stabilizes the generation of Th17 cells. The transcription factors of Th17 cells [retinoid-related orphan receptor (ROR) γt, ROR-α and signal transducer and activator of transcription-3] have been described recently. Since TGF-β is essential for the generation of both Th17 and regulatory T (Treg) cells from naive T cells, which suggests a developmental link between Th17 and Treg cells. Functions of these two subsets of T cells are, however, opposite to each other; Th17 cells are highly pathogenic during the inflammatory process while Treg cells are crucial for inhibiting tissue inflammation and maintaining self-tolerance. Here, we review the recent information on differentiation and effector functions of Th17 cells during inflammatory conditions. [ABSTRACT FROM AUTHOR]

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive Th cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4+ T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-γ and had no effect on IL-4 expression. In parallel to the predominant release of the Th1 cytokine IFN-γ, up-regulation of CCR5 on the immune CD4+ T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1α and regulated on activation normal T cell expressed and secreted showed Th1 bias. The porin-primed CD4+ T cells expressed the mRNA for T-box expressed in T cells, a Th1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector Th1 cells. The immune CD4+ T cells proliferated and released IL-2 and IFN-γ profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4+ T cells of Th1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity. [ABSTRACT FROM AUTHOR]

The immunoscope methodology has proven useful to analyze T-cell repertoires in mice and humans. We adapted it to the analysis of VH chains of human peripheral B cells by setting up a quantification of various VH and JH segments and the profiling of IgM-, IgG-, IgA- and IgE-expressing B cells. We then tested the hypothesis that the human B-cell and T-cell repertoires have a similar diversity of VH and V-beta rearrangements. We studied in more detail the VH5 family because it is not abundantly used, which facilitated the analysis. The data showed that the number of distinct VH5 rearrangements in all samples studied is close to the number of cells in the sample. This contrasts with T cells in which we previously showed that distinct V-beta rearrangements amount to a few percent of the number of T cells because each V-beta chain is on the average paired with ∼25 alpha chains. Thus, in the VH5 family, the light chains add little quantitative diversity to that produced by the heavy chain alone. Whether this feature can be generalized to other VH chains is discussed. [ABSTRACT FROM AUTHOR]