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1.
Proc Natl Acad Sci U S A ; 120(52): e2301155120, 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-38109544

ABSTRACT

The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell-dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.


Subject(s)
Caspases , Lymphoma, Large B-Cell, Diffuse , Mice , Humans , Animals , Adult , Tensins/genetics , Caspases/metabolism , NF-kappa B/metabolism , Cell Adhesion/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/genetics , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , Lymphoma, Large B-Cell, Diffuse/genetics , Integrins
2.
Nat Immunol ; 24(5): 869-883, 2023 05.
Article in English | MEDLINE | ID: mdl-37081150

ABSTRACT

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.


Subject(s)
CD8-Positive T-Lymphocytes , Immunotherapy, Adoptive , Interleukin-2 , Neoplasms, Experimental , CD8-Positive T-Lymphocytes/immunology , T-Cell Exhaustion , Lymphocytes, Tumor-Infiltrating/immunology , Interleukin-2/pharmacology , Interleukin-33 , Protein Engineering , Female , Animals , Mice , Mice, Inbred C57BL , Cell Line, Tumor , Neoplasms, Experimental/therapy , Programmed Cell Death 1 Receptor/metabolism
3.
Front Cell Infect Microbiol ; 12: 941860, 2022.
Article in English | MEDLINE | ID: mdl-36034709

ABSTRACT

The lymphatic system plays a crucial role in mounting immune response against intracellular pathogens, and recent studies have documented its role in facilitating tumor dissemination linked largely with cancer cells. However, in mucocutaneous leishmaniasis (MCL) caused by Leishmania Viannia subgenus showing infectious metastasis and resulting in severe distant secondary lesions, the route of escape of these parasites to secondary sites has not yet been investigated in detail. Our results demonstrated that when infection was associated with inflammation and additionally exacerbated by the presence of dsRNA viral endosymbiont (LRV1), lymphatic vessels could serve as efficient routes for infected cells to egress from the primary site and colonize distant organs. We challenged this hypothesis by using the intracellular Leishmania protozoan parasites Leishmania guyanensis (Lgy) associated with or without a dsRNA viral endosymbiont, exacerbating the infection and responsible for a strong inflammatory response, and favoring metastasis of the infection. We analyzed possible cargo cells and the routes of dissemination through flow cytometry, histological analysis, and in vivo imaging in our metastatic model to show that parasites disseminated not only intracellularly but also as free extracellular parasites using migrating immune cells, lymph nodes (LNs), and lymph vessels, and followed intricate connections of draining and non-draining lymph node to finally end up in the blood and in distant skin, causing new lesions.


Subject(s)
Leishmania braziliensis , Leishmania , Leishmaniasis, Mucocutaneous , Neoplasms , Humans , Lymphatic System
4.
Immunity ; 53(5): 985-1000.e11, 2020 11 17.
Article in English | MEDLINE | ID: mdl-33128876

ABSTRACT

Central memory CD8+ T cells (Tcm) control systemic secondary infections and can protect from chronic infection and cancer as a result of their stem-cell-like capacity to expand, differentiate, and self-renew. Central memory is generally thought to emerge following pathogen clearance and to form based on the de-differentiation of cytolytic effector cells. Here, we uncovered rare effector-phase CD8+ T cells expressing high amounts of the transcription factor Tcf7 (Tcf1) that showed no evidence of prior cytolytic differentiation and that displayed key hallmarks of Tcm cells. These effector-phase Tcf7hi cells quantitatively yielded Tcm cells based on lineage tracing. Mechanistically, Tcf1 counteracted the differentiation of Tcf7hi cells and sustained the expression of conserved adult stem-cell genes that were critical for CD8+ T cell stemness. The discovery of stem-cell-like CD8+ T cells during the effector response to acute infection provides an opportunity to optimize Tcm cell formation by vaccination.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Cytotoxicity, Immunologic , Hepatocyte Nuclear Factor 1-alpha/metabolism , Immunologic Memory , T Cell Transcription Factor 1/metabolism , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation/genetics , Chromatin Assembly and Disassembly , Cytotoxicity, Immunologic/genetics , Fluorescent Antibody Technique , Gene Expression , Hepatocyte Nuclear Factor 1-alpha/chemistry , Hepatocyte Nuclear Factor 1-alpha/genetics , Humans , Immunization , Immunologic Memory/genetics , Immunophenotyping , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Molecular , Protein Conformation , Spleen/immunology , Spleen/metabolism , Structure-Activity Relationship , T Cell Transcription Factor 1/chemistry , T Cell Transcription Factor 1/genetics
5.
Immunity ; 50(1): 195-211.e10, 2019 01 15.
Article in English | MEDLINE | ID: mdl-30635237

ABSTRACT

Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8+ T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1+PD-1+ TILs mediated the proliferative response to immunotherapy, generating both Tcf1+PD-1+ and differentiated Tcf1-PD-1+ cells. Ablation of Tcf1+PD-1+ TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1+PD-1+ TILs but was essential for the stem-like functions of these cells. Human TCF1+PD-1+ cells were detected among tumor-reactive CD8+ T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.


Subject(s)
Antibodies, Monoclonal/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Stem Cells/immunology , T-Lymphocyte Subsets/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , Cell Differentiation , Cell Proliferation , Hepatitis A Virus Cellular Receptor 2/antagonists & inhibitors , Hepatocyte Nuclear Factor 1-alpha/genetics , Humans , Immunotherapy , Lymphocytes, Tumor-Infiltrating/drug effects , Melanoma/immunology , Melanoma, Experimental , Mice , Mice, Inbred C57BL
6.
Proc Natl Acad Sci U S A ; 115(29): E6826-E6835, 2018 07 17.
Article in English | MEDLINE | ID: mdl-29967180

ABSTRACT

Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread.


Subject(s)
Antibody Formation , Homeostasis/immunology , Lymph Nodes/immunology , Plasma Cells/immunology , Animals , B-Cell Activating Factor/immunology , Chemokine CXCL12/immunology , Interleukin-6/immunology , Lymph Nodes/cytology , Male , Mice , Plasma Cells/cytology , Stromal Cells/cytology , Stromal Cells/immunology
7.
J Clin Invest ; 127(4): 1574-1588, 2017 Apr 03.
Article in English | MEDLINE | ID: mdl-28319044

ABSTRACT

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.


Subject(s)
Graft vs Host Disease/immunology , Lymph Nodes/pathology , Receptors, Notch/physiology , Spleen/pathology , T-Lymphocytes/immunology , Allografts , Animals , Bone Marrow Transplantation , Calcium-Binding Proteins , Cells, Cultured , Female , Fibroblasts/immunology , Graft vs Host Disease/metabolism , Graft vs Host Disease/pathology , Intercellular Signaling Peptides and Proteins/physiology , Ligands , Lymph Nodes/immunology , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Spleen/immunology , T-Lymphocytes/metabolism
8.
J Immunol ; 197(3): 771-82, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27324132

ABSTRACT

Although Notch signaling plays important roles in lineage commitment and differentiation of multiple cell types including conventional T cells, nothing is currently known concerning Notch function in innate-like T cells. We have found that the homeostasis of several well-characterized populations of innate-like T cells including invariant NKT cells (iNKT), CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells is controlled by Notch. Notch selectively regulates hepatic iNKT cell survival via tissue-restricted control of B cell lymphoma 2 and IL-7Rα expression. More generally, Notch regulation of innate-like T cell homeostasis involves both cell-intrinsic and -extrinsic mechanisms and relies upon context-dependent interactions with Notch ligand-expressing fibroblastic stromal cells. Collectively, using conditional ablation of Notch receptors on peripheral T cells or Notch ligands on putative fibroblastic stromal cells, we show that Notch signaling is indispensable for the homeostasis of three tissue-restricted populations of innate-like T cells: hepatic iNKT, CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells, thus supporting a generalized role for Notch in innate T cell homeostasis.


Subject(s)
Cell Differentiation/immunology , Homeostasis/immunology , Receptors, Notch/immunology , Signal Transduction/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Animals , Flow Cytometry , Immunohistochemistry , Mice , Mice, Transgenic , Receptors, Notch/metabolism
9.
Proc Natl Acad Sci U S A ; 111(1): E109-18, 2014 Jan 07.
Article in English | MEDLINE | ID: mdl-24367096

ABSTRACT

Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.


Subject(s)
Fibroblasts/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocytes/cytology , Adaptive Immunity , Animals , Cell Movement , Cell Proliferation , Dendritic Cells/cytology , Fibroblasts/metabolism , Homeostasis , Inflammation , Lymphocyte Activation , Lymphotoxin-alpha/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Myeloid Differentiation Factor 88/metabolism , Stromal Cells/cytology , T-Lymphocytes/cytology
10.
Genesis ; 51(10): 717-24, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23832856

ABSTRACT

The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium.


Subject(s)
Epithelial Cells/metabolism , Forkhead Transcription Factors/genetics , Gene Expression , Mice, Transgenic , Models, Animal , Thymus Gland/embryology , Animals , Cells, Cultured , Chromosomes, Artificial, Bacterial , Epithelium/embryology , Epithelium/metabolism , Female , Gene Expression Regulation, Developmental , Mice , Mice, Inbred C57BL , Organ Specificity , Promoter Regions, Genetic , Thymus Gland/metabolism
11.
PLoS One ; 6(11): e27618, 2011.
Article in English | MEDLINE | ID: mdl-22110693

ABSTRACT

Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/-) mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.


Subject(s)
Fibroblasts/cytology , Fibroblasts/metabolism , Lymph Nodes/cytology , Nitric Oxide/biosynthesis , T-Lymphocytes/cytology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Dendritic Cells/cytology , Dendritic Cells/immunology , Gene Expression Regulation, Enzymologic/immunology , Interferon-gamma/metabolism , Mice , Nitric Oxide Synthase Type II/deficiency , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
12.
Methods Mol Biol ; 612: 313-23, 2010.
Article in English | MEDLINE | ID: mdl-20033650

ABSTRACT

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.


Subject(s)
Gene Expression Regulation , NK Cell Lectin-Like Receptor Subfamily A/metabolism , Animals , Cell Adhesion , Cell Line , Cell Membrane/metabolism , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Ligands , Protein Binding , Solubility , Substrate Specificity
13.
Immunity ; 31(4): 598-608, 2009 Oct 16.
Article in English | MEDLINE | ID: mdl-19818651

ABSTRACT

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/metabolism , NK Cell Lectin-Like Receptor Subfamily A/chemistry , NK Cell Lectin-Like Receptor Subfamily A/metabolism , Animals , Histocompatibility Antigens Class I/immunology , Immunological Synapses/immunology , Immunological Synapses/metabolism , Killer Cells, Natural/immunology , Mice , Mice, Inbred C3H , NK Cell Lectin-Like Receptor Subfamily A/immunology , Protein Binding/immunology , Protein Conformation , Protein Multimerization
14.
Immunity ; 30(3): 337-47, 2009 Mar 20.
Article in English | MEDLINE | ID: mdl-19249231

ABSTRACT

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily A/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Flow Cytometry , Genetic Variation , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A/genetics
15.
Blood ; 111(7): 3571-8, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18198346

ABSTRACT

NKG2D is a multisubunit activation receptor that allows natural killer (NK) cells to detect and eliminate stressed, infected, and transformed host cells. However, the chronic exposure of NK cells to cell-bound NKG2D ligands has been shown to impair NKG2D function both in vitro and in vivo. Here we have tested whether continuous NKG2D engagement selectively impacted NKG2D function or whether heterologous NK cell activation pathways were also affected. We found that sustained NKG2D engagement induced cross-tolerization of several unrelated NK cell activation receptors. We show that receptors that activate NK cells via the DAP12/KARAP and DAP10 signaling adaptors, such as murine NKG2D and Ly49D, cross-tolerize preferentially NK cell activation pathways that function independent of DAP10/12, such as antibody-dependent cell-mediated cytotoxicity and missing-self recognition. Conversely, DAP10/12-independent pathways are unable to cross-tolerize unrelated NK cell activation receptors such as NKG2D or Ly49D. These data define a class of NK cell activation receptors that can tolerize mature NK cells. The reversible suppression of the NK cells' cytolytic function probably reduces the NK cells' efficacy to control endogenous and exogenous stress yet may be needed to limit tissue damage.


Subject(s)
Immune Tolerance/physiology , Killer Cells, Natural/immunology , Lymphocyte Activation/physiology , Receptors, Immunologic/immunology , Adaptor Proteins, Signal Transducing/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/physiology , Antigens, Ly/immunology , Lectins, C-Type/immunology , Ligands , Membrane Proteins/immunology , Mice , NK Cell Lectin-Like Receptor Subfamily A , NK Cell Lectin-Like Receptor Subfamily K , Receptors, Immunologic/antagonists & inhibitors , Receptors, NK Cell Lectin-Like , Receptors, Natural Killer Cell
16.
Blood ; 111(1): 142-9, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17906078

ABSTRACT

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin.


Subject(s)
Cell Lineage/physiology , Hematopoiesis/physiology , Thymus Gland/cytology , beta Catenin/genetics , gamma Catenin/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hybridomas , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction/physiology , Spleen/cytology , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , gamma Catenin/metabolism
17.
Proc Natl Acad Sci U S A ; 104(10): 3978-83, 2007 Mar 06.
Article in English | MEDLINE | ID: mdl-17360463

ABSTRACT

Ly49A is an inhibitory receptor, which counteracts natural killer (NK) cell activation on the engagement with H-2D(d) (D(d)) MHC class I molecules (MHC-I) on target cells. In addition to binding D(d) on apposed membranes, Ly49A interacts with D(d) ligand expressed in the plane of the NK cells' membrane. Indeed, multivalent, soluble MHC-I ligand binds inefficiently to Ly49A unless the NK cells' D(d) complexes are destroyed. However, it is not known whether masked Ly49A remains constitutively associated with cis D(d) also during target cell interaction. Alternatively, it is possible that Ly49A has to be unmasked to significantly interact with its ligand on target cells. These two scenarios suggest distinct roles of Ly49A/D(d) cis interaction for NK cell function. Here, we show that Ly49A contributes to target cell adhesion and efficiently accumulates at synapses with D(d)-expressing target cells when NK cells themselves lack D(d). When NK cells express D(d), Ly49A no longer contributes to adhesion, and ligand-driven recruitment to the cellular contact site is strongly reduced. The destruction of D(d) complexes on NK cells, which unmasks Ly49A, is necessary and sufficient to restore Ly49A adhesive function and recruitment to the synapse. Thus, cis D(d) continuously sequesters a considerable fraction of Ly49A receptors, preventing efficient Ly49A recruitment to the synapse with D(d)+ target cells. The reduced number of Ly49A receptors that can functionally interact with D(d) on target cells explains the modest inhibitory capacity of Ly49A in D(d) NK cells. This property renders Ly49A NK cells more sensitive to react to diseased host cells.


Subject(s)
Antigens, Ly/chemistry , Antigens, Ly/genetics , H-2 Antigens/chemistry , Killer Cells, Natural/cytology , Animals , Bacterial Proteins/metabolism , Cytotoxicity, Immunologic , Flow Cytometry , Genes, MHC Class I , Histocompatibility Antigen H-2D , Ligands , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , Receptors, Immunologic/metabolism , Synapses/metabolism
18.
J Immunol ; 178(3): 1277-84, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17237373

ABSTRACT

The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells' own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells' own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.


Subject(s)
Antigens, Ly/metabolism , Histocompatibility Antigens Class I/metabolism , Lectins, C-Type/metabolism , Animals , Killer Cells, Natural/immunology , Ligands , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A , Protein Binding , Receptors, Immunologic/metabolism , Receptors, NK Cell Lectin-Like
19.
Blood ; 106(5): 1726-33, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-15890681

ABSTRACT

Intrathymic T-cell maturation critically depends on the selective expansion of thymocytes expressing a functionally rearranged T-cell receptor (TCR) beta chain. In addition, TCR-independent signals also contribute to normal T-cell development. It is unclear whether and how signals from the 2 types of pathways are integrated. Here, we show that T-cell factor-1 (TCF-1), a nuclear effector of the canonical wingless/int (wnt)/catenin signaling pathway, ensures the survival of proliferating, pre-TCR(+) thymocytes. The survival of pre-TCR(+) thymocytes requires the presence of the N-terminal catenin-binding domain in TCF-1. This domain can bind the transcriptional coactivator beta-catenin and may also bind gamma-catenin (plakoglobin). However, in the absence of gamma-catenin, T-cell development is normal, supporting a role for beta-catenin. Signaling competent beta-catenin is present prior to and thus arises independently from pre-TCR signaling and does not substantially increase on pre-TCR signaling. In contrast, pre-TCR signaling significantly induces TCF-1 expression. This coincides with the activation of a wnt/catenin/TCF reporter transgene in vivo. Collectively, these data suggest that efficient TCF-dependent transcription requires that pre-TCR signaling induces TCF-1 expression, whereas wnt signals may provide the coactivator such as beta-catenin. The 2 pathways thus have to cooperate to ensure thymocyte survival at the pre-TCR stage.


Subject(s)
DNA-Binding Proteins/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Transcription Factors/immunology , Animals , Cell Cycle/drug effects , Cell Cycle/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Survival/drug effects , Cell Survival/immunology , Cytoskeletal Proteins/pharmacology , DNA-Binding Proteins/deficiency , Desmoplakins , Hepatocyte Nuclear Factor 1-alpha , Homeodomain Proteins/immunology , Intercellular Signaling Peptides and Proteins/immunology , Lymphoid Enhancer-Binding Factor 1 , Mice , Mice, Inbred C57BL , Structure-Activity Relationship , T Cell Transcription Factor 1 , Thymus Gland/cytology , Thymus Gland/immunology , Transcription Factors/deficiency , Wnt Proteins , gamma Catenin
20.
Nat Immunol ; 5(3): 328-36, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14973437

ABSTRACT

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.


Subject(s)
Antigens, Ly/physiology , Cytotoxicity, Immunologic , Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/immunology , Receptors, Immunologic/physiology , Animals , Antigens, Ly/genetics , Antigens, Ly/metabolism , Binding Sites , Cell Line , Cell Line, Tumor , Cytotoxicity Tests, Immunologic , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Lectins, C-Type , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Immunological , Mosaicism , NK Cell Lectin-Like Receptor Subfamily A , Neoplasms/immunology , Receptors, Immunologic/metabolism , Receptors, NK Cell Lectin-Like , Transfection
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