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1.
Genome Biol Evol ; 8(11): 3364-3376, 2016 12 14.
Article in English | MEDLINE | ID: mdl-27979968

ABSTRACT

The RAG recombinase is a domesticated transposable element co-opted in jawed vertebrates to drive the process of the so-called V(D)J recombination, which is the hallmark of the adaptive immune system to produce antigen receptors. RAG targets, namely, the Recombination Signal Sequences (RSS), are rather long and degenerated sequences, which highlights the ability of the recombinase to interact with a wide range of target sequences, including outside of antigen receptor loci. The recognition of such cryptic targets by the recombinase threatens genome integrity by promoting aberrant DNA recombination, as observed in lymphoid malignancies. Genomes evolution resulting from RAG acquisition is an ongoing discussion, in particular regarding the counter-selection of sequences resembling the RSS and the modifications of epigenetic regulation at these potential cryptic sites. Here, we describe a new bioinformatics tool to map potential RAG targets in all jawed vertebrates. We show that our REcombination Classifier (REC) outperforms the currently available tool and is suitable for full genomes scans from species other than human and mouse. Using the REC, we document a reduction in density of potential RAG targets at the transcription start sites of genes co-expressed with the rag genes and marked with high levels of the trimethylation of the lysine 4 of the histone 3 (H3K4me3), which correlates with the retention of functional RAG activity after the horizontal transfer.


Subject(s)
Evolution, Molecular , Nucleotide Motifs , Selection, Genetic , VDJ Recombinases/genetics , Animals , Genome , Sequence Analysis, DNA/methods , Software , Vertebrates/genetics
2.
Mucosal Immunol ; 9(3): 787-97, 2016 05.
Article in English | MEDLINE | ID: mdl-26487367

ABSTRACT

Deregulation of host-microbiota interactions in the gut is a pivotal characteristic of Crohn's disease. It remains unclear, however, whether commensals and/or the dysbiotic microbiota associated with pathology in humans are causally involved in Crohn's pathogenesis. Here, we show that Crohn's-like ileitis in Tnf(ΔARE/+) mice is microbiota-dependent. Germ-free Tnf(ΔARE/+) mice are disease-free and the microbiota and its innate recognition through Myd88 are indispensable for tumor necrosis factor (TNF) overexpression and disease initiation in this model. The epithelium of diseased mice shows no major defects in mucus barrier and paracellular permeability. However, Tnf(ΔARE/+) ileitis associates with the reduction of lysozyme-expressing Paneth cells, mediated by adaptive immune effectors. Furthermore, we show that established but not early ileitis in Tnf(ΔARE/+) mice involves defective expression of antimicrobials and dysbiosis, characterized by Firmicutes expansion, including epithelial-attaching segmented filamentous bacteria, and decreased abundance of Bacteroidetes. Microbiota modulation by antibiotic treatment at an early disease stage rescues ileitis. Our results suggest that the indigenous microbiota is sufficient to drive TNF overexpression and Crohn's ileitis in the genetically susceptible Tnf(ΔARE/+) hosts, whereas dysbiosis in this model results from disease-associated alterations including loss of lysozyme-expressing Paneth cells.


Subject(s)
Crohn Disease/immunology , Dysbiosis/immunology , Gastrointestinal Microbiome/immunology , Ileitis/immunology , Intestinal Mucosa/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Crohn Disease/microbiology , Disease Models, Animal , Dysbiosis/microbiology , Host-Pathogen Interactions , Humans , Ileitis/microbiology , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Tumor Necrosis Factor-alpha/genetics
3.
Clin Exp Immunol ; 182(3): 251-60, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26206289

ABSTRACT

Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery.


Subject(s)
Diabetes Mellitus, Experimental/therapy , Diabetes Mellitus, Type 1/therapy , Immunotherapy , Peptides/pharmacology , Proinsulin/pharmacology , T-Lymphocytes, Regulatory/immunology , Animals , Autoantibodies/immunology , Cell Proliferation/drug effects , Cell Proliferation/genetics , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/pathology , HLA-DR4 Antigen/genetics , HLA-DR4 Antigen/immunology , HLA-DRB1 Chains/genetics , HLA-DRB1 Chains/immunology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Mice , Mice, Transgenic , T-Lymphocytes, Regulatory/pathology
4.
Scand J Immunol ; 74(6): 585-95, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21916921

ABSTRACT

It is well established that viral, parasitic or bacterial infections can prevent type 1 diabetes (T1D) occurrence in non-obese diabetic (NOD) mice. On the other hand, defects in CD4(+) Regulatory T cell (Treg) numbers and/or function contribute to T1D aetiology in NOD mice and in humans. In this work, we formally tested whether the protective role of the bacterial product lipopolysaccharide (LPS) on diabetes incidence results from enhanced Treg activity. We first report that weekly administration of LPS to young prediabetic NOD mice, presenting or not insulitis at the time of treatment, afforded full protection from diabetes. Taking advantage from the high but incomplete penetrance of diabetes in NOD mice raised in specific pathogen free (SPF) conditions we compared untreated disease-free old animals with gender- and age-matched LPS-treated mice. Histological and flow cytometry analysis indicated that LPS treatment did not prevent islet infiltration or priming of diabetogenic T cells but increased Foxp3(+) and CD103(+) Treg frequency and numbers. By performing adoptive transfer experiments into alymphoid NOD/SCID recipients, we further demonstrated that CD25(+) cells from LPS-treated NOD mice, but not from naturally protected animals, maintained diabetogenic cells at check. Our study suggests that T cell regulation represents a cellular mechanism to explain the 'hygiene hypothesis' and reinforces the notion that immune activity consolidates dominant tolerance.


Subject(s)
Diabetes Mellitus, Type 1/immunology , Lipopolysaccharides/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigens, CD/immunology , Female , Forkhead Transcription Factors/immunology , Integrin alpha Chains/immunology , Mice , Mice, Inbred NOD
5.
Genes Immun ; 10(1): 93-9, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18818690

ABSTRACT

Natural IgM are involved in numerous immunological functions but the genetic factors that control the homeostasis of its secretion and upholding remain unknown. Prompted by the finding that C57BL/6 mice had significantly lower serum levels of IgM when compared with BALB/c mice, we performed a genome-wide screen and found that the level of serum IgM was controlled by a QTL on chromosome 13 reaching the highest level of association at marker D13Mit266 (LOD score=3.54). This locus was named IgMSC1 and covered a region encompassing the interferon-regulatory factor 4 gene (Irf4). The number of splenic mature B cells in C57BL/6 did not differ from BALB/c mice but we found that low serum levels of IgM in C57BL/6 mice correlated with lower frequency of IgM-secreting cells in the spleen and in the peritoneal cavity. These results suggested that C57BL/6 mice have lower efficiency in late B-cell maturation, a process that is highly impaired in Irf4 knockout mice. In fact, we also found reduced Irf4 gene expression in B cells of C57BL/6 mice. Thus, we propose Irf4 as a candidate for the IgMSC1 locus, which controls IgM homeostatic levels at the level of B-cell terminal differentiation.


Subject(s)
Homeostasis , Immunoglobulin M/blood , Immunoglobulin M/genetics , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Chromosomes, Mammalian , Genetic Markers , Immunoglobulin M/immunology , Immunoglobulin M/metabolism , Interferon Regulatory Factors/immunology , Lod Score , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peritoneal Cavity/cytology , Quantitative Trait Loci , Spleen/cytology , Spleen/immunology , Spleen/metabolism
6.
Curr Top Microbiol Immunol ; 293: 43-71, 2005.
Article in English | MEDLINE | ID: mdl-15981475

ABSTRACT

The seminal work of Le Douarin and colleagues (Ohki et al. 1987; Ohki et al. 1988; Salaun et al. 1990; Coutinho et al. 1993) first demonstrated that peripheral tissue-specific tolerance is centrally established in the thymus, by epithelial stromal cells (TEC). Subsequent experiments have shown that TEC-tolerance is dominant and mediated by CD4 regulatory T cells (Treg) that are generated intrathymically by recognition of antigens expressed on TECs (Modigliani et al. 1995; Modigliani et al. 1996a). From these and other observations, in 1996 Modigliani and colleagues derived a general model for the establishment and maintenance of natural tolerance (MM96) (Modigliani et al. 1996b), with two central propositions: (1) T cell receptor (TCR)-dependent sorting of emergent repertoires generates TEC-specific Treg displaying the highest TCR self-affinities below deletion thresholds, thus isolating repertoires undergoing positive and negative selection; (2) Treg are intrathymically committed (and activated) for a unique differentiative pathway with regulatory effector functions. The model explained the embryonic/perinatal time window of natural tolerance acquisition, by developmental programs determining (1) TCR multireactivity, (2) the cellular composition in the thymic stroma (relative abundance of epithelial vs hemopoietic cells), and (3) the dynamics of peripheral lymphocyte pools, built by accumulation of recent thymic emigrants (RTE) that remain recruitable to regulatory functions. We discuss here the MM96 in the light of recent results demonstrating the promiscuous expression of tissue-specific antigens by medullary TECs (Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004) and indicating that Treg represent a unique differentiative pathway (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003), which is adopted by CD4 T cells with high avidity for TEC-antigens (Bensinger et al. 2001; Jordan et al. 2001; Apostolou et al. 2002). In the likelihood that autoimmune diseases (AID) result from Treg deficits, some of which might have a thymic origin, we also speculate on therapeutic strategies aiming at selectively stimulating their de novo production or peripheral function, within recent findings on Treg responses to inflammation (Caramalho et al. 2003; Lopes-Carvalho et al., submitted, Caramalho et al., submitted). In short, the MM96 argued that natural tolerance is dominant, established and maintained by the activity of Treg, which are selected upon high-affinity recognition of self-ligands on TECs, and committed intrathymically to a unique differentiative pathway geared to anti-inflammatory and antiproliferative effector functions. By postulating the intrathymic deletion of self-reactivities on hemopoietic stromal cells (THC), together with the inability of peripheral resident lymphocytes to engage in the regulatory pathway, the MM96 simultaneously explained the maintenance of responsiveness to non-self in a context of suppression mediating dominant self-tolerance. The major difficulty of the MM96 is related to the apparent tissue specificity of Treg repertoires generated intrathymically. This difficulty has now been principally solved by the work of Hanahan, Kyewski and others (Jolicoeur et al. 1994; Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004), demonstrating the selective expression of a variety of tissue-specific antigens by TECs, in topological patterns that are compatible with the MM96, but difficult to conciliate with recessive tolerance models (Kappler et al. 1987; Kisielow et al. 1988). While the developmentally regulated multireactivity of TCR repertoires (Gavin and Bevan 1995), as well as the peripheral recruitment of Treg among RTE (Modigliani et al. 1996a) might add to this process, it would seem that the establishment of tissue-specific tolerance essentially stems from the "promiscuous expression of tissue antigens" by TEC. The findings of AID resulting from natural mutations (reviewed in Pitkanen and Peterson 2003) or the targeted inactivation (Anderson et al. 2002; Ramsey et al. 2002) of the AIRE transcription factor that regulates promiscuous gene expression on TECs support this conclusion. The observations on the correlation of natural or forced expression of the Foxp3 transcription factor in CD4 T cells with Treg phenotype and function (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003) provided support for the MM96 contention that Treg represent a unique differentiative pathway that is naturally established inside the thymus. Furthermore, Caton and colleagues (Jordan et al. 2001), as well as several other groups (Bensinger et al. 2001; Apostolou et al. 2002), have provided direct evidence for our postulate that Treg are selected among differentiating CD4 T cells with high affinity for ligands expressed on TECs (Modigliani et al. 1996b). Finally, the demonstration by Caramalho et al. that Treg express innate immunity receptors (Caramalho et al. 2003) and respond to pro-inflammatory signals and products of inflammation (Caramalho et al., submitted) brought about a new understanding on the peripheral regulation of Treg function. Together with the observation that Treg also respond to ongoing activities of "naïve/effector" T cells--possibly through the IL-2 produced in these conditions--these findings explain the participation of Treg in all immune responses (Onizuka et al. 1999; Shimizu et al. 1999; Annacker et al. 2001; Curotto de Lafaille et al. 2001; Almeida et al. 2002; Shevach 2002; Bach and Francois Bach 2003; Wood and Sakaguchi 2003; Mittrucker and Kaufmann 2004; Sakaguchi 2004), beyond their fundamental role in ensuring self-tolerance (e.g., Modigliani et al. 1996a; Shevach 2000; Hori et al. 2003; Sakaguchi 2004; Thompson and Powrie 2004). Thus, anti-inflammatory and anti-proliferative Treg are amplified by signals that promote or mediate inflammation and proliferation, accounting for the quality control of responses (Coutinho et al. 2001). In turn, such natural regulation of Treg by immune responses to non-self may well explain the alarming epidemiology of allergic and AID in wealthy societies (Wills-Karp et al. 2001; Bach 2002; Yazdanbakhsh et al. 2002), where a variety of childhood infections have become rare or absent. Thus, it is plausible that Treg were evolutionarily set by a given density of infectious agents in the environment. With hindsight, it is not too surprising that natural Treg performance falls once hygiene, vaccination, and antibiotics suddenly (i.e., 100 years) plunged infectious density to below some critical physiological threshold. As the immune system is not adapted to modern clean conditions of postnatal development, clinical immunologists must now deal with frequent Treg deficiencies (allergies and AID) for which they have no curative or rational treatments. It is essential, therefore, that basic immunologists concentrate on strategies to selectively stimulate the production, survival, and activity of this set of lymphocytes that is instrumental in preventing immune pathology. We have argued that the culprit of this inability of basic research to solve major clinical problems has been the self-righteousness of recessive tolerance champions, from Ehrlich to some of our contemporaries. It is ironical, however, that none of us--including the heretic opponents of horror autotoxicus--had understood that self-tolerance, or its robustness at least, is in part determined by the frequency and intensity of the responses to non-self. In the evolution of ideas on immunological tolerance, the time might be ripe for some kinds of synthesis. First, conventional theory reduced self-tolerance to negative selection and microbial defense to positive selection, while the MM96 solution was the precise opposite: positive selection of autoreactivities for self-tolerance (Treg) and negative selection (of Treg) for ridding responses. In contrast, it would now appear that positive and negative selection of autoreactive T cells are both necessary to establish either self-tolerance or competence to eliminate microbes, two processes that actually reinforce each other in the maintenance of self-integrity. Second, V-region recognition has generally been held responsible for specific discrimination between what should be either tolerated or eliminated from the organism. In contrast again, it would now seem that both processes of self-tolerance and microbial defense (self/non-self discrimination) also operate on the basis of evolutionarily ancient, germ-line-encoded innate, nonspecific receptors (Medzhitov and Janeway 2000) capable of a coarse level of self/non-self discrimination (Coutinho 1975). It could thus be interesting to revisit notions of cooperativity between V-regions and such mitogen receptors, both in single cell functions (Coutinho et al. 1974) and in the system's evolution (Coutinho 1975, 1980) as well. After all, major transitions in evolution were cooperative (Maynard-Smith and Szathmary 1995).


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Receptors, Interleukin-2/immunology , Self Tolerance , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Autoimmune Diseases/immunology , Biological Evolution , Humans , Lymphopoiesis , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology
7.
Eur J Immunol ; 31(12): 3763-72, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11745397

ABSTRACT

The recombination activating proteins (RAG1 and RAG2) are essential for V(D)J recombination of immunoglobulin chains. Expression of both genes is lymphocyte-specific and RAG levels are tightly regulated throughout lymphopoiesis and cell cycle. To assess the significance of this pattern of expression, we generated transgenic mice expressing the Rag genes both continuously throughout lymphocyte development and constitutively in most non-lymphoid tissues. The transgenes partially complement an endogenous Rag2 null mutation and lead to a partial block in early B and T lymphopoiesis when introduced on a Rag2 sufficient background. The defect in thymocyte number is restricted to the alpha beta lineage leaving the gamma delta T cell pool intact, while neither IgH phenotypic allelic exclusion nor the kappa/lambda light chain ratio are altered. Finally, the ectopic expression of the Rag genes associates with growth retardation and early death of the animals, a phenotype reminiscent of those reported for mice deficient in double-strand break repair molecules.


Subject(s)
DNA-Binding Proteins/genetics , Genes, RAG-1/physiology , Lymphopenia/etiology , Alleles , Animals , Growth Disorders/etiology , Immunoglobulin Heavy Chains/genetics , Lymphocyte Count , Mice , Mice, Inbred C57BL , Mice, Transgenic , Transgenes
8.
Immunol Rev ; 182: 89-98, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11722626

ABSTRACT

Little progress has been achieved over the last 20 years on the clinical management of several conditions that relate to self-tolerance and to the regulation of immune responses: autoimmune diseases, transplantation tolerance, tumor immunity, allergy and vaccine development in chronic infections. These failures, it is argued, are due to the inability of the prevalent "recessive tolerance" concepts to accommodate physiological autoreactivity and the regulatory potential it embodies. In this review, the advantages of "dominant tolerance" models are underlined in the light of critical evidence and in the general context of the natural autoimmune activities. The role of regulatory T cells is discussed, notably in the regulation of inflammatory reactions and, more generally, in the "quality control" of immune responses. It is anticipated that progress will be brought about by dominant tolerance approaches, and through an increased knowledge of the differentiative pathways, repertoires, mechanisms of activation and effector functions of autoreactive, regulatory T cells.


Subject(s)
Autoimmunity/immunology , Self Tolerance/immunology , T-Lymphocytes/immunology , Animals , Autoimmune Diseases/immunology , Humans , Hypersensitivity/immunology , Inflammation Mediators/immunology , Neoplasms/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Transplantation Immunology
9.
J Exp Med ; 194(8): 1141-50, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11602642

ABSTRACT

Interleukin 7 is a crucial factor for the development of murine T and B lymphocytes. We now report that, in the absence of interleukin 7, B lymphocyte production takes place exclusively during fetal and perinatal life, ceasing after 7 wk of age. In peripheral organs, however, the pool of B lymphocytes is stable throughout adult life and consists only of cells that belong to the B1 and marginal zone (MZ) compartments. This is accompanied by a 50-fold increase in the frequency of immunoglobulin (Ig)M- and IgG-secreting cells, and the concentration of serum immunoglobulins is increased three- to fivefold. Both the MZ phenotype and the increase in serum IgM are T cell independent. These findings reveal a previously undescribed pathway of B lymphopoiesis that is active in early life and is interleukin 7 independent. This pathway generates B1 cells and a normal sized MZ B lymphocyte compartment.


Subject(s)
B-Lymphocytes/cytology , Interleukin-7/immunology , Lymphocyte Activation/immunology , Aging/blood , Animals , B-Lymphocytes/immunology , Biomarkers , Bone Marrow Cells/cytology , Cell Differentiation , Female , Hematopoiesis , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Immunophenotyping , Interleukin-7/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Plasma Cells/cytology , Plasma Cells/immunology , Spleen/cytology
10.
Int Immunol ; 11(2): 279-88, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10069426

ABSTRACT

Locally produced type I interferon (IFN-I) enhances the sensitivity of bone marrow B cell to IgM receptor ligation. The establishment of B cell repertoires, on the other hand, seems to involve selective processes that are critically dependent on B cell receptor (BCR) ligation. In order to assess the importance of BCR triggering thresholds on the selection of polyclonal unmanipulated B cell populations, we compared VH gene expression and reactivity repertoires in various B cell compartments of wild-type and IFN-I receptor-deficient mice (IFN-I-R-/-). These analyses demonstrate that increased B cell sensitivity to BCR ligation mediated by IFN-I in the bone marrow (BM) has consequences on the stringency of B cell repertoire selection. Thus, the normal counter-selection of both VH7183 gene family expression and multireactivity was impaired among immature BM B cells from mutant mice. Furthermore, as a result of reduced efficiency of BCR ligation-dependent inhibition of terminal differentiation, IFN-I-R-/- animals produce, in BM and thymus, higher numbers of plasma cells secreting antibodies that are more multireactive than wild-type animals. Finally, mutant serum IgM natural antibodies display a more reactive repertoire than controls, a likely reflection of the BM resident plasma cell repertoire. The present observations demonstrate, therefore, that local modulation of BCR triggering thresholds leads to important modifications in the generation and/or selection of normal B cell populations.


Subject(s)
Antibody Formation , B-Lymphocytes/immunology , Bone Marrow Cells/immunology , Interferon Type I/immunology , Animals , Cell Differentiation , Enzyme-Linked Immunosorbent Assay , Genes, Immunoglobulin , Immunoglobulin G/blood , Immunoglobulin M/blood , In Situ Hybridization , Mice , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolism
11.
Int Immunol ; 9(5): 755-62, 1997 May.
Article in English | MEDLINE | ID: mdl-9184921

ABSTRACT

Ligation of surface IgM on B cells responding to lipopolysaccharide (LPS) suppresses terminal differentiation and high-rate Ig secretion with no effect on proliferation. As shown here, different B cell populations show characteristic mean values of ligand concentration required for 50% inhibition, with Gaussian distributions of sensitivity to IgM receptor ligation that reflect cellular heterogeneity of 'al-or-none' inhibitions in single cells. Differential sensitivity of B cell populations to IgM ligation seems to be locally determined by the cellular environment and unrelated to the 'maturity' of the responding cells. Thus, while long-lived peritoneal B cells are 3- to 5-fold more resistant than splenic B cells, there is no difference in sensitivity between short- and long-lived B cells in the spleen. Furthermore, while B cells in bone marrow and spleen differ in sensitivity by two orders of magnitude, B cells differentiated in vitro from bone marrow pre-B cells are as resistant as splenic B cells. Moreover, bone marrow cell culture supernatants restore a high level of sensitivity in such cell populations. Differential sensitivity to IgM receptor ligation is reproduced by multivalent nominal antigen, in cell populations that show identical dose-response inhibition curves to direct activation of protein kinase C by phorbol esters. We conclude that the level of sensitivity to IgM ligation is independent of the life span or maturity of the B cell, but differentially regulated in vivo by putative tissue factors.


Subject(s)
B-Lymphocyte Subsets/metabolism , Immunoglobulin M/metabolism , Receptors, Fc/metabolism , Animals , Animals, Newborn , Antibodies, Anti-Idiotypic/pharmacology , B-Lymphocyte Subsets/drug effects , B-Lymphocyte Subsets/immunology , Bone Marrow/immunology , Bone Marrow/metabolism , Cell Survival/immunology , Cells, Cultured , Immunoglobulin M/immunology , Immunosuppressive Agents/pharmacology , Ligands , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Models, Immunological , Receptors, Antigen, B-Cell/metabolism , Sensitivity and Specificity , Solubility , Spleen/immunology , Tetradecanoylphorbol Acetate/pharmacology
12.
Int Immunol ; 9(11): 1677-85, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9418129

ABSTRACT

Compartmentation of B lymphocyte populations is associated with differences in both development stage and sensitivity to Ig (sIg)-dependent triggering. In order to characterize the factors that contribute in setting the level of sensitivity of a B cell, we quantified sIgM-dependent regulation of Ig secretion in purified mature and immature B cells after ex vivo and in vivo modification of their environment. These analyses formally demonstrate that the bone marrow (BM) microenvironment locally induces high B cell sensitivity to sIgM ligation irrespective of differentiation stage. We further provide evidence that BM macrophages create a dominant environment that enhances B cell sensitivity to B cell receptor triggering. Finally, using ex vivo assays as well as type I IFN receptor-deficient mice we show that IFN-beta produced by resident BM macrophages is necessary and sufficient to define B cell sensitivity. Implications of these findings for the understanding of B cell selection processes are discussed.


Subject(s)
B-Lymphocytes/physiology , Interferon-beta/physiology , Macrophages/metabolism , Receptors, Fc/physiology , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Differentiation/physiology , Immunoglobulin M/physiology , Interferon-beta/biosynthesis , Mice , Mice, Inbred C57BL , Sensitivity and Specificity
13.
Proc Natl Acad Sci U S A ; 93(15): 7871-6, 1996 Jul 23.
Article in English | MEDLINE | ID: mdl-8755569

ABSTRACT

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.


Subject(s)
DNA-Binding Proteins , Enhancer Elements, Genetic , Gene Deletion , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Receptors, Antigen, T-Cell, alpha-beta/genetics , Alleles , Animals , B-Lymphocytes/immunology , Base Sequence , Blastocyst , Cells, Cultured , Chimera , DNA Nucleotidyltransferases/metabolism , DNA Primers , Genetic Complementation Test , Genomic Library , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Joining Region/biosynthesis , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Proteins/genetics , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Restriction Mapping , Sequence Deletion , Spleen/immunology , Stem Cells , Transcription, Genetic , VDJ Recombinases
14.
Int Immunol ; 7(12): 1995-2003, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8746569

ABSTRACT

The accessibility of a chromosomally integrated TCR beta minilocus recombination substrate in a V(D)J recombinase-inducible cell line (HDR37) depends on incorporation of transcriptional enhancer elements such as the Ig kappa light chain intronic enhancer (E kappa). The E kappa element contains several functional motifs including the kappa B motif, which binds the NF-kappa B transcription factor. To assess molecular mechanisms by which E kappa promotes V(D)J recombinational accessibility, we compared the abilities of the wild-type E kappa, a corresponding E kappa sequence with a mutant kappa B motif (E kappa-kappa B-) and a kappa B motif dimer (kappa B2) to function in the context of the TCR beta minilocus/HDR37 system. The E kappa-containing minilocus underwent demethylation, transcription and V(D)J recombination, independently of copy number of integration site. Transfectants containing low copy numbers (one or two) of the E kappa-kappa B(-)-containing minilocus, like enhancerless or kappa B2-containing miniloci at any copy number, were inactive with respect to all three processes. In contrast, high-copy-number integrants of the E kappa-kappa B- substrates showed an integration-site dependent activation of all three processes. Together these data show that the kappa B motif plays a critical role in the ability of E kappa to confer V(D)J recombinational accessibility, but that it is not sufficient to mediate this process by itself.


Subject(s)
Immunoglobulin kappa-Chains/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Recombination, Genetic , Animals , Base Sequence , Cell Line , DNA/chemistry , DNA/genetics , DNA Nucleotidyltransferases/metabolism , DNA Primers/genetics , Enhancer Elements, Genetic , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Introns , Methylation , Mice , Molecular Sequence Data , Transfection , VDJ Recombinases
15.
Int Immunol ; 7(10): 1637-47, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8562509

ABSTRACT

Myc family proteins are thought to be transcription factors involved in regulation of cell growth and differentiation. N-myc is expressed at the pre-B cell stage of B cell differentiation and is dramatically induced by the pre-B cell growth factor, IL-7. To test the idea that N-myc plays an important role in lymphocyte development, we assayed the effect of a null N-myc mutation on the differentiation of B and T lineage cells. Homozygous, mutant embryonic stem (ES) cells were injected into blastocysts derived from recombination activating gene (RAG-2)-deficient mice. Since RAG-2 mutant mice fail to develop mature lymphocytes, later-stage lymphocytes that are present in chimeric mice are ES cell derived. Surprisingly, nearly normal numbers of mature T and B cells derived from N-myc-deficient ES cells were found in peripheral lymphoid organs of chimeric mice. Lymphocytes were judged to be functional based on responses to mitogens and production of serum IgM and multiple IgG isotypes in chimeric animals. We discuss these findings in relation to N-myc function in lymphocyte development and possible redundancy with other myc genes.


Subject(s)
DNA-Binding Proteins , Genes, myc , Hematopoiesis/genetics , Lymphocytes/cytology , Proteins/physiology , Proto-Oncogene Proteins c-myc/physiology , Stem Cells/cytology , Animals , Blastocyst , Cell Differentiation , Cell Lineage , Chimera , Genes, Lethal , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Leukemia Virus, Murine/physiology , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-myc/deficiency
16.
Cell ; 80(5): 813-23, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7889575

ABSTRACT

Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.


Subject(s)
DNA Repair/genetics , DNA-Binding Proteins , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombination, Genetic/genetics , Severe Combined Immunodeficiency/genetics , Amino Acid Sequence , Animals , Base Sequence , CHO Cells/radiation effects , Cell Extracts/chemistry , Chromosome Mapping , Chromosomes, Artificial, Yeast/genetics , Cricetinae , DNA/metabolism , DNA-Activated Protein Kinase , Gamma Rays , Genetic Complementation Test , Immunoglobulin Variable Region/genetics , Mice , Mice, SCID , Molecular Sequence Data , Radiation Tolerance/genetics
17.
Science ; 265(5177): 1442-5, 1994 Sep 02.
Article in English | MEDLINE | ID: mdl-8073286

ABSTRACT

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.


Subject(s)
Antigens, Nuclear , DNA Helicases , DNA Repair/genetics , DNA-Binding Proteins/genetics , Genes, Immunoglobulin , Nuclear Proteins/genetics , Receptors, Antigen, T-Cell/genetics , Recombination, Genetic , Animals , Base Sequence , CHO Cells , Cell Survival/radiation effects , Cloning, Molecular , Cricetinae , DNA Damage , DNA-Binding Proteins/metabolism , Genetic Complementation Test , Humans , Hybrid Cells , Ku Autoantigen , Molecular Sequence Data , Nuclear Proteins/metabolism , Transfection
18.
J Virol ; 64(11): 5633-9, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2170689

ABSTRACT

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.


Subject(s)
Polyomavirus/growth & development , Tumor Virus Infections/microbiology , Virus Replication , Animals , Bone and Bones/microbiology , DNA, Viral/analysis , Female , Male , Mammary Glands, Animal/microbiology , Mice , Mice, Nude , Nucleic Acid Hybridization , Salivary Glands/microbiology , Sex Factors , Skin/microbiology
20.
Genomics ; 7(4): 587-93, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2387585

ABSTRACT

The murine and human genes for the L1 neural adhesion molecule were shown to lie on conserved regions of the X chromosome to which genes responsible for several neuromuscular diseases have been mapped and which are adjacent to the fragile site (FRAXA) associated with mental retardation. By pulsed-field gel mapping we have demonstrated physical linkage between the L1 gene and other genes located in Xq28: L1 lies between the eye pigment RCP, GCP locus and the glucose-6-phosphate dehydrogenase (G6PD) gene. This location is compatible with the implication of the L1 molecule in one of the X-linked neuromuscular diseases mapped to this region.


Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Genetic Linkage , X Chromosome , Amino Acid Sequence , Animals , Base Sequence , Chromosome Banding , Chromosome Mapping , Fragile X Syndrome/genetics , Genes , Guanosine Diphosphate/genetics , Humans , Immunoglobulins/genetics , Mice , Molecular Sequence Data , Multigene Family , Nucleic Acid Hybridization , Restriction Mapping , Retinal Pigments/genetics , Sequence Homology, Nucleic Acid
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