Surface Cytotoxic T Lymphocyte–associated Antigen 4 Partitions Within Lipid Rafts and Relocates to the Immunological Synapse under Conditions of Inhibition of T Cell Activation

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR–CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4–mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation

Preclinical evaluation of cancer immune therapy using patient-derived tumor antigen-specific T cells in a novel xenograft platform.

Objectives: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel Methods: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD- Results: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells. Conclusion: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies

Viewpoint: Therapeutic Implications of CTLA-4 Compartmentalization

Understanding the regulatory events involved in the activation and inactivation of T cells is crucial to develop therapeutic approaches for autoimmune diseases and for organ transplantation. Co-stimulatory signals delivered through the CD28 receptor and inhibitory signals through CTLA-4 are required for the proper modulation of T cell responses and the induction and maintenance of peripheral tolerance. Manipulation of these signals is emerging as a potential strategy to prevent allograft rejection in different animal models. Recent data on the compartmentalization and the structural features of CTLA-4 within T cells provides critical information not only on the molecular basis of T cell inactivation by CTLA-4, but also on the key requirements for the successful development of therapeutic strategies targeting this molecule

Specific CD3{varepsilon} Epsilon Association of a Phosphodiesterase 4B Isoform Determines Its Selective Tyrosine Phosphorylation after CD3 Ligation

cAMP-specific phosphodiesterases (PDE) comprise an extensive family of enzymes that control intracellular levels of cAMP and thus regulate T cell responses. It is not known how the function of these enzymes is altered by TCR engagement. We have examined this issue by studying one of the PDE isozymes (PDE4B). PDE4B RNA and protein were detected in resting PBLs, and the levels of PDE4B protein increased with cell cycling. In peripheral blood T cells, two previously reported PDE4B isoforms could be detected: one was 75–80 kDa (PDE4B1) and the other was 65–67 kDa (PDE4B2). These two isoforms differed in their N-terminal sequence, with the presence of four potential myristylation sites in the PDE4B2 that are absent in PDE4B1. Consequently, only PDE4B2 was found in association with the CD3{varepsilon} chain of the TCR. In addition, although both isoforms were phosphorylated in tyrosines in pervanadate-stimulated T cells, only the TCR-associated PDE4B2 was tyrosine-phosphorylated following CD3 ligation. The kinetics of phosphorylation of TCR-associated PDE4B2 correlated with changes in cAMP levels, suggesting that tyrosine phosphorylation of the TCR-associated PDE4B isoform upon engagement of this receptor may be an important regulatory step in PDE4B function. Our results reveal that selectivity of PDE4B activation can be achieved by differential receptor association and phosphorylation of the alternatively spliced forms of this PDE

Abnormal Immunological Profile and Vaginal Microbiota in Women Prone to Urinary Tract Infections▿

The host determinants of susceptibility to recurrent urinary tract infections (UTI) are poorly understood. We investigated whether the susceptibility is associated with abnormalities in the immunological defense and further explored the linkage to vaginal microbiota. For this purpose, we compared vaginal, urine, and blood samples collected during a disease-free period from 22 women with recurrent UTI and from 17 controls. In UTI-prone women, interleukin-12 (IL-12) production in peripheral monocytes and myeloid dendritic cells (DCs) was significantly (P < 0.05) enhanced whether measured in relative numbers of IL-12-producing cells or in mean IL-12 production per cell. In contrast, no T-cell polarization was observed. Interestingly, it seemed that the cytokine production of DCs and monocytes did not translate into T-cell activation in the UTI-prone group in a manner similar to that seen with the controls. In vaginal mucosa, UTI-prone women had a lower concentration of tissue repair-associated vascular endothelial growth factor (VEGF) (P = 0.006) and less often had detectable amounts of the chief monocyte and DC chemoattractant, monocyte chemotactic protein 1 (P = 0.005), than the controls. The microbiota of UTI-prone women was characterized by a diminished lactobacillus morphotype composition, with an abnormally high (>3) mean Nugent score of 4.6 compared to 1.7 for the controls (P = 0.003). Normal lactobacillus composition was associated with increased IL-17 and VEGF concentrations in vaginal mucosa. In conclusion, immunological defects and a persistently aberrant microbiota, a lack of lactobacilli in particular, may contribute to susceptibility to recurrent UTI. Further studies of antigen-presenting-cell function and T-cell activation in recurrent UTI are called for

Detection of a lactobacillus substance that inhibits Escherichia coli

The host determinants of susceptibility to recurrent urinary tract infections (UTI) are poorly understood. We investigated whether the susceptibility is associated with abnormalities in the immunological defense and further explored the linkage to vaginal microbiota. For this purpose, we compared vaginal, urine, and blood samples collected during a disease-free period from 22 women with recurrent UTI and from 17 controls. In UTI-prone women, interleukin-12 (IL-12) production in peripheral monocytes and myeloid dendritic cells (DCs) was significantly (P \u3c 0.05) enhanced whether measured in relative numbers of IL-12-producing cells or in mean IL-12 production per cell. In contrast, no T-cell polarization was observed. Interestingly, it seemed that the cytokine production of DCs and monocytes did not translate into T-cell activation in the UTI-prone group in a manner similar to that seen with the controls. In vaginal mucosa, UTI-prone women had a lower concentration of tissue repair-associated vascular endothelial growth factor (VEGF) (P = 0.006) and less often had detectable amounts of the chief monocyte and DC chemoattractant, monocyte chemotactic protein 1 (P = 0.005), than the controls. The microbiota of UTI-prone women was characterized by a diminished lactobacillus morphotype composition, with an abnormally high (\u3e3) mean Nugent score of 4.6 compared to 1.7 for the controls (P = 0.003). Normal lactobacillus composition was associated with increased IL-17 and VEGF concentrations in vaginal mucosa. In conclusion, immunological defects and a persistently aberrant microbiota, a lack of lactobacilli in particular, may contribute to susceptibility to recurrent UTI. Further studies of antigen-presenting-cell function and T-cell activation in recurrent UTI are called for. Copyright © 2009, American Society for Microbiology. All Rights Reserved

Oral Administration of the Probiotic Combination Lactobacillus Rhamnosus GR-1 and L. Fermentum RC-14 for Human Intestinal Applications

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14, previously characterized as urogenital probiotics were evaluated for human intestinal applications. RC-14 and GR-1 were tolerant to 0.3 and 0.5% (w/v) bile, respectively. Both strains were suspended in skim milk, stored as a frozen concentrate and administered in combination to five healthy women twice daily for 14 days. Faecal samples were analyzed and the Lactobacillus flora assessed by Randomly Amplified Polymorphic DNA (RAPD). Both strains were recovered from all subjects during the 14-day administration period and GR-1 was detected for at least 7 days post-administration in some individuals. No notable increases in serum IgG, IgA or IgM were observed and IL-2 and IL-4 were undetectable. Although IL-6 and IFN-γ levels increased slightly in some individuals, concentrations remained within normal ranges. Thus, L. rhamnosus GR-1 and L. fermentum RC-14 are bile tolerant and survive gastrointestinal transit without induction of systemic immune or inflammatory responses. These data together with the previously demonstrated probiotic properties of GR-1 and RC-14 make this strain combination potentially useful for human intestinal applications

Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 to prevent or cure bacterial vaginosis among women with HIV

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14, previously characterized as urogenital probiotics were evaluated for human intestinal applications. RC-14 and GR-1 were tolerant to 0.3 and 0.5% (w/v) bile, respectively. Both strains were suspended in skim milk, stored as a frozen concentrate and administered in combination to five healthy women twice daily for 14 days. Faecal samples were analyzed and the Lactobacillus flora assessed by Randomly Amplified Polymorphic DNA (RAPD). Both strains were recovered from all subjects during the 14-day administration period and GR-1 was detected for at least 7 days post-administration in some individuals. No notable increases in serum IgG, IgA or IgM were observed and IL-2 and IL-4 were undetectable. Although IL-6 and IFN-γ levels increased slightly in some individuals, concentrations remained within normal ranges. Thus, L. rhamnosus GR-1 and L. fermentum RC-14 are bile tolerant and survive gastrointestinal transit without induction of systemic immune or inflammatory responses. These data together with the previously demonstrated probiotic properties of GR-1 and RC-14 make this strain combination potentially useful for human intestinal applications. © 2002 Elsevier Science Ltd. All rights reserved

The Inhibitory Function of CTLA-4 Does Not Require Its Tyrosine Phosphorylation

CTLA-4 is a negative regulator of T cell responses. Sequence analysis of this molecule reveals the presence of two cytoplasmic tyrosine residues at positions 165 and 182 that are potential Src homology (SH)-2 domain binding sites. The role of phosphorylation of these residues in CTLA-4-mediated signaling is unknown. Here, we show that sole TCR ligation induces zeta-associated protein (ZAP)-70-dependent tyrosine phosphorylation of CTLA-4 that is important for cell surface retention of this molecule. However, CTLA-4 tyrosine phosphorylation is not required for down-regulation of T cell activation following CD3-CTLA-4 coengagement. Specifically, inhibition of extracellular signal-regulated kinase (ERK) activation and of IL-2 production by CTLA-4-mediated signaling occurs in T cells expressing mutant CTLA-4 molecules lacking the cytoplasmic tyrosine residues, and in lck-deficient or ZAP-70-deficient T cells. Therefore, CTLA-4 function involves interplay between two different levels of regulation: phosphotyrosine-dependent cell surface retention and phosphotyrosine-independent association with signaling molecules

Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation