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1.  Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice 
PLoS ONE  2012;7(12):e52161.
Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220+ CD4–CD8– (DN) T lymphocytes. The precise ontogeny of B220+ DN T cells is unknown. B220+ DN T lymphocytes could be derived from effector CD4+ and CD8+ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220+ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220+ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220+ T cells observed in normal MRL+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220+ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.
PMCID: PMC3528777  PMID: 23284917
2.  P2 receptors and immunity 
Immune cells express receptors for extracellular nucleotides named P2 receptors. P2 receptors transduce signals delivered by nucleotides present in the extracellular environment. Accruing evidence shows that purinergic signalling has a profound effect on multiple immune cell responses such as T lymphocyte proliferation, chemotaxis, cytokine release, phagocytosis, Ag presentation and cytotoxicity. This makes P2 receptors an attractive target for the therapy of immuno-mediated disease and cancer.
PMCID: PMC3514633  PMID: 22909902
Nucleotides; Adenosine triphosphate; Damage associated molecular patterns (DAMP); Purinergic receptors; Inflammasome; Autoimmune diseases
3.  Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis 
European journal of immunology  2011;41(6):1696-1708.
Activated B cells can regulate immunity, and have been envisaged as potential cell-based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered to express antigens in a remarkably simple, rapid, and effective way with lentiviral vectors. Notably, this neither required nor induced activation of the B cells. With that approach we were able to produce reprogrammed resting B cells that inhibited antigen-specific CD4+ T cells, CD8+ T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in experimental autoimmune encephalomyelitis, and even induced complete remission from disease in mice lacking functional natural regulatory T cells, which otherwise developed a chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity.
PMCID: PMC3431508  PMID: 21469107
B cells; autoimmunity; gene therapy; lentiviral vector; cellular therapy
4.  Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection 
The Journal of Experimental Medicine  2011;208(9):1823-1834.
Contact with HIV-1 envelope protein elicits release of ATP through pannexin-1 channels on target cells; by activating purinergic receptors and Pyk2 kinase in target cells, this extracellular ATP boosts HIV-1 infectivity.
Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.
PMCID: PMC3171090  PMID: 21859844
5.  The human and mouse orthologous LIM-only proteins respectively encoded in chromosome 6 and 17 show a different expression pattern 
Thymocytes interact with various subpopulations of thymic epithelial cells (TECs) at different stages of their development. To identify new molecules specifically expressed in TECs and/or thymic nurse cells (TNCs), we used representational difference analysis. We identified a LIM protein located on mouse chromosome 17 (m17TLP) and belonging to the family of the LIM-only proteins (LIMo). We found a new splice variant in addition to the two describedA and B isoforms. The three alternative species of m17TLP are found strictly in the thymic stroma. This protein is expressed on a subpopulation of TECs and TNCs. Strikingly, we found that the human ortholog of m17TLP, located on chromosome 6 (h6LIMo), is expressed in most tissues, but not in skeletal muscle. We have identified four human splice variants of h6LIMo which differ in their carboxy-terminal regions. The sequence comprising the genomic structure suggests that CRP2 is the closest known relative of m17TLP. Although the human and mouse nucleotide sequences are 88–97% homologous, this homology is reduced to 47% in the promoter regions, which strongly suggests that their differential expression is related to their promoter regulatory activity.
PMCID: PMC2778486  PMID: 15380775
LIM-only protein; LIM domains; Thymus; Representational difference analysis; Thymic epithelial cells; Thymic nurse cells
6.  An affinity/avidity model of peripheral T cell regulation 
Journal of Clinical Investigation  2005;115(2):302-312.
We show in these studies that Qa-1–dependent CD8+ T cells are involved in the establishment and maintenance of peripheral self tolerance as well as facilitating affinity maturation of CD4+ T cells responding to foreign antigen. We provide experimental evidence that the strategy used by the Qa-1–dependent CD8+ T cells to accomplish both these tasks in vivo is to selectively downregulate T cell clones that respond to both self and foreign antigens with intermediate, not high or low, affinity/avidity. Thus, the immune system evolved to regulate peripheral immunity using a unified mechanism that efficiently and effectively permits the system to safeguard peripheral self tolerance yet promote the capacity to deal with foreign invaders.
PMCID: PMC544609  PMID: 15668735
7.  Most α/β T Cell Receptor Diversity Is Due to Terminal Deoxynucleotidyl Transferase 
The Journal of Experimental Medicine  2001;194(9):1385-1390.
The contribution of template-independent nucleotide addition to antigen receptor diversity is unknown. We therefore determined the size of the T cell receptor (TCR)α/β repertoire in mice bearing a null mutation on both alleles of the terminal deoxynucleotidyl transferase (Tdt) gene. We used a method based upon polymerase chain reaction amplification and exhaustive sequencing of various AV-AJ and BV-BJ combinations. In both wild-type and Tdt°/° mice, TCRAV diversity is one order of magnitude lower than the TCRBV diversity. In Tdt°/° animals, TCRBV chain diversity is reduced 10-fold compared with wild-type mice. In addition, in Tdt°/° mice, one BV chain can associate with three to four AV chains as in wild-type mice. The α/β repertoire size in Tdt°/° mice is estimated to be 105 distinct receptors, ∼5–10% of that calculated for wild-type mice. Thus, while Tdt activity is not involved in the combinatorial diversity resulting from α/β pairing, it contributes to at least 90% of TCRα/β diversity.
PMCID: PMC2195970  PMID: 11696602
T cell repertoire; T cell receptor; knockout mice; terminal deoxynucleotidyl transferase; CDR3
8.  Interleukin 4–Producing Cd4 T Cells Arise from Different Precursors Depending on the Conditions of Antigen Exposure in Vivo 
The precursor origin of T helper (Th) cell subsets in vivo has been difficult to study and remains poorly investigated. We have previously shown that chronic administration of soluble protein antigen induces selective development of antigen-specific CD4 Th2 cells in genetically predisposed mouse strains. To analyze the origin of effector T cells in this model, we designed a competitive polymerase chain reaction–based approach to track public BV-J rearrangement expressed by CD4 T cells specific for hen egg white lysozyme (HEL) in BALB/c mice. We show that public T cell clones are predominantly associated with type 1 or 2 effector Th cells recovered after primary immunization in complete or incomplete Freund's adjuvant, respectively. Conversely, continuous administration of soluble antigen, which induces strong memory Th2 response, is associated with a dose-dependent reduction of public clone size by a mechanism resembling clonal anergy. Thus, soluble HEL–induced Th2 cells do not express the public complementarity determining region 3 motifs characteristic of immunogenic challenge in the presence of adjuvant. These results demonstrate that there are multiple pathways of induction of Th2 responses depending on the condition of antigen exposure in vivo, i.e., clonal immune deviation versus recruitment of a different pool of precursor cells.
PMCID: PMC2195842  PMID: 10684860
clonal expansion; antigen-specific public repertoire; CD4 T cell subsets; chronic antigen stimulation; clonal anergy
9.  Quantitative Analysis of the T Cell Repertoire Selected by a Single Peptide–Major Histocompatibility Complex  
The Journal of Experimental Medicine  1998;187(11):1871-1883.
The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide–MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vβ11-Jβ1.1 or Vβ12-Jβ1.1 rearrangements. We found that a single peptide–MHC class II complex positively selects at least 105 different Vβ rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Eα52-68–I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the β chain repertoire bears the imprint of the selecting self-peptide.
PMCID: PMC2212317  PMID: 9607927
thymus; major histocompatibility complex; T cell receptors; repertoire development; transgenic/knockout
10.  A New Marker on Chicken Hematopoietic Cells is Defined by a Monoclonal Antibody Raised Against a V ß Chain of the Human TCR 
Developmental Immunology  1992;2(4):273-284.
In this paper, we show that a mouse monoclonal antibody, 111-427, specific for the V ß 5.3 chain of the human T-cell receptor (TCR) for antigen, also reacts with chicken hematopoietic cells. Our data indicate that the majority of 111-427 positive cells among peripheral blood leucocytes (PBL) are thrombocytes. This antibody also recognizes two in vitro cell lines, III-C5, an IL-2-dependent T-cell-line and HD11, a macrophage cell line. In addition, erythrocytes and a minor subpopulation of thymus and spleen cells are also stained by the monoclonal antibody (mAb). No specific immunoprecipitation could be detected from 125I radiolabeled cell lysates. By Western blotting techniques, the 111- 427 mAb identifies a single band of apparent molecular weight 91 kD, unaffected by reduction, from III-C5 and HD11 cell lysates. This band is absent in negative cell control lysates. On thrombocytes, the apparent molecular weight of the band is shifted to 87 kD. These results indicate that the mAb does not recognize the chicken T-cell receptor for antigen, but a cell surface marker shared primarily between thrombocytes and erythrocytes. This new chicken cell marker is compared to other cell surface markers in avian or mammalian species that present some analogies in their tissue distribution.
PMCID: PMC2275868  PMID: 1343096
Cross-reactive monoclonal antibody; T-cell receptor; chicken; hematopoietic cells; thrombocytes; erythrocytes

Results 1-10 (10)