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ABSTRACT: Interactions between TCR and self-peptide/MHC complex play an important role in homeostasis and Ag reactivity of mature peripheral T cells. In this report, we demonstrate that the interactions between mature peripheral T cells and endogenous Ags have a negative impact on the maintenance of foreign Ag-specific T cells in an age-dependent manner. This is mediated by RAG-dependent secondary rearrangement of the TCR alpha-chain (receptor revision). The TCR revision in mature T cells is readily observed in mouse expressing transgenic TCR alpha-chain inserted into the physiological locus (knockin mouse) but not in conventional transgenic mouse with an identical TCR alpha-chain. Thus, our results suggest that under physiological conditions in which all TCR alpha-chains are susceptible to deletion by secondary rearrangement, TCR revision in mature peripheral T cells is an ongoing process in adult animals and contributes to age-dependent changes in T cell function and repertoire.
The Journal of Immunology 09/2007; 179(4):2163-9. · 5.79 Impact Factor
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ABSTRACT: CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.
The Journal of Immunology 08/2007; 179(2):1122-8. · 5.79 Impact Factor
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ABSTRACT: Immunoglobulin E (IgE) induces mast cell survival in the absence of antigen (Ag) through the high-affinity IgE receptor, Fcepsilon receptor I (FcepsilonRI). Although we have shown that protein tyrosine kinase Syk and sustained extracellular signal-regulated kinase (Erk) activation are required for IgE-induced mast cell survival, how Syk couples with sustained Erk activation is still unclear. Here, we report that the transmembrane adaptors LAT and NTAL are phosphorylated slowly upon IgE stimulation and that sustained but not transient Erk activation induced by IgE was inhibited in LAT(-/-) NTAL(-/-) bone marrow-derived mast cells (BMMCs). IgE-induced survival requires Ras activation, and both were impaired in LAT(-/-) NTAL(-/-) BMMCs. Sos was preferentially required for FcepsilonRI signals by IgE rather than IgE plus Ag. Survival impaired in LAT(-/-) NTAL(-/-) BMMCs was restored to levels comparable to those of the wild type by membrane-targeted Sos, which bypasses the Grb2-mediated membrane recruitment of Sos. The IgE-induced survival of BMMCs lacking Gads, an adaptor critical for the formation of the LAT-SLP-76-phospholipase Cgamma (PLCgamma) complex, was observed to be normal. IgE stimulation induced the membrane retention of Grb2-green fluorescent protein fusion proteins in wild-type but not LAT(-/-) NTAL(-/-) BMMCs. These results suggest that LAT and NTAL contribute to the maintenance of Erk activation and survival through the membrane retention of the Ras-activating complex Grb2-Sos and, further, that the LAT-Gads-SLP-76-PLCgamma and LAT/NTAL-Grb2-Sos pathways are differentially required for degranulation and survival, respectively.
Molecular and Cellular Biology 07/2007; 27(12):4406-15. · 5.53 Impact Factor
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Ana V Miletic,
Kumiko Sakata-Sogawa,
Michio Hiroshima,
Michael J Hamann,
Timothy S Gomez,
Naruhisa Ota,
Tracie Kloeppel, Osami Kanagawa,
Makio Tokunaga,
Daniel D Billadeau,
Wojciech Swat
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ABSTRACT: Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.
Journal of Biological Chemistry 01/2007; 281(50):38257-65. · 4.77 Impact Factor
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ABSTRACT: Incomplete T cell antigen receptor-mediated signaling induces an unresponsive state known as anergy. Previously, we had shown that anergy can be induced in antigen-primed but not naive T cells. In this report, we found that in vitro primed T cells from IL-2R alpha-deficient mice were resistant to anergy induction in contrast to comparably treated wild-type T cells. This resistance persisted even after proliferation of IL-2R alpha chain-deficient CD4 T cells with high-dose IL-2-IL-2R beta gamma chains interaction. Thus, antigen activation, and/or progression through cell cycle are not sufficient to induce anergy susceptibility in T cells. The high-affinity IL-2-IL-2R interaction appears to play a critical role in this process.
International Immunology 06/2006; 18(5):645-51. · 3.41 Impact Factor
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ABSTRACT: To become mature alphabeta T cells, developing thymocytes must first assemble a T cell receptor (TCR) beta chain gene encoding a TCRbeta chain that forms a pre-TCR. These cells then need to generate a TCRalpha chain gene encoding a TCRalpha chain, which, when paired with the TCRbeta chain, forms a selectable alphabeta TCR. Newly generated VJalpha rearrangements that do not encode TCRalpha chains capable of forming selectable alphabeta TCRs can be excised from the chromosome and replaced with new VJalpha rearrangements. Such replacement occurs through the process of TCRalpha chain gene revision whereby a Valpha gene segment upstream of the VJalpha rearrangement is appended to a downstream Jalpha gene segment. A multistep, gene-targeting approach was used to generate a modified TCRalpha locus (TCRalpha(sJ)) with a limited capacity to undergo revision of TCRalpha chain genes. Thymocytes from mice homozygous for the TCRalpha(sJ) allele are defective in their ability to generate an alphabeta TCR. Furthermore, those thymocytes that do generate an alphabeta TCR have a diminished capacity to be positively selected, and TCRalpha(sJ/sJ) mice have significantly reduced numbers of mature alphabeta T cells. Together, these findings demonstrate that normal T cell development relies on the ability of developing thymocytes to revise their TCRalpha chain genes.
Proceedings of the National Academy of Sciences 11/2005; 102(40):14356-61. · 9.68 Impact Factor
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ABSTRACT: CD4(-)CD8(-) thymocytes expressing a transgenic T cell receptor (TCR) alpha chain have decreased capacity to give rise to CD4(+)CD8(+) thymocytes when compared with wild-type thymocytes. This inefficient CD4(-)CD8(-) to CD4(+)CD8(+) maturation is mediated by the transgenic TCR alpha chain pairing with endogenous TCR beta chain but not with endogenous TCR gamma chain. Comparison between TCR alpha chain-transgenic mice with or without a functional pre-TCR alpha (pT alpha ) chain reveals that the formation of transgenic alpha/endogenous beta TCR on CD4(-)CD8(-) thymocytes inhibits the formation of pre-TCR, but at the same time mediates CD4(-)CD8(-) to CD4(+)CD8(+) maturation in the absence of pre-TCR, albeit inefficiently. These results indicate that alpha beta TCR and pre-TCR provide different signals for thymocyte development. They also suggest that the precise regulation of the sequential rearrangements of TCR beta and alpha loci and the cellular expansion induced by the pre-TCR may both be evolved to ensure the efficient generation of mature alpha beta T cells.
European Journal of Immunology 07/2004; 34(6):1532-41. · 5.10 Impact Factor
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ABSTRACT: Diabetogenic BDC2.5 CD4 T cells induce diabetes when injected into NOD.scid mice. However, when co-transferred with the OVA-specific DO11.10 CD4 T cells, BDC2.5 T cells failed to cause diabetes. This inhibition depended upon the stimulation of DO11.10 T cells only with soluble OVA, which skewed their differentiation to a Th2-type pattern of cytokine secretion in vivo. However, in vivo neutralization of IL-4, IL-10 or TGF-beta using monoclonal antibodies did not prevent the inhibition whereas treatment with an antibody against the glucocorticoid-induced TNF receptor abrogated the protection from disease. In the protected mice, the diabetogenic T cells could be isolated from their spleens and shown to transfer diabetes when injected into new NOD.scid recipients. Thus, the inhibition took place without the physical or functional elimination of the diabetogenic T cells.
European Journal of Immunology 03/2004; 34(2):447-54. · 5.10 Impact Factor
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ABSTRACT: We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic beta-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-A(g7), and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse beta cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic beta cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a beta cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.
International Immunology 01/2004; 15(12):1473-83. · 3.41 Impact Factor
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Kyeong-Hee Lee,
Aaron R Dinner,
Chun Tu,
Gabriele Campi,
Subhadip Raychaudhuri,
Rajat Varma,
Tasha N Sims,
W Richard Burack,
Hui Wu,
Julia Wang, Osami Kanagawa,
Mary Markiewicz,
Paul M Allen,
Michael L Dustin,
Arup K Chakraborty,
Andrey S Shaw
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ABSTRACT: The immunological synapse is a specialized cell-cell junction between T cell and antigen-presenting cell surfaces. It is characterized by a central cluster of antigen receptors, a ring of integrin family adhesion molecules, and temporal stability over hours. The role of this specific organization in signaling for T cell activation has been controversial. We use in vitro and in silico experiments to determine that the immunological synapse acts as a type of adaptive controller that both boosts T cell receptor triggering and attenuates strong signals.
Science 12/2003; 302(5648):1218-22. · 31.20 Impact Factor
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ABSTRACT: The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.
European Journal of Immunology 07/2003; 33(6):1568-75. · 5.10 Impact Factor
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ABSTRACT: We isolated and identified naturally processed peptides selected by antigen-presenting cells homozygous for expression of I-A(g7) or I-A(d) class II MHC molecules, or from heterozygous antigen-presenting cells that express I-A(g7) along with I-A(g7PD) or I-A(d). Identification of large numbers of peptides demonstrated that despite being closely related on a structural level, each class II MHC molecule selected for very unique peptides. The large data sets allowed us to definitively establish the preferred peptide-binding motifs critical for selection of peptides by I-A(g7), I-A(g7PD), and I-A(d). Finally, extensive analyses of peptide families reveals that there was little competition among class II MHC alleles for display of peptides and that presence of one allele had minimal impact on the repertoire of peptides selected by another.
Proceedings of the National Academy of Sciences 05/2003; 100(9):5330-5. · 9.68 Impact Factor
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ABSTRACT: Interferon-producing cells (IPCs) secrete high levels of type I interferon in response to certain viruses. The lack of lineage markers, the expression of major histocompatibility complex (MHC) class II and the capacity to stimulate allogeneic T cells have led these cells to be classified as a subset of dendritic cells (DCs), called plasmacytoid DCs (PDCs). However, the role of IPCs/PDCs in initiating primary immune responses remains elusive. Here we examined the antigen presenting capacity of murine IPCs in antigen specific systems. While CD8alpha+ and CD11b+ DCs induced logarithmic expansion of naive CD4 and CD8 T cells, without conferring T helper commitment at a first encounter, primary IPCs lacked the ability to stimulate naive T cells. However, when antigen-experienced, nonpolarized T cells expanded by classical DC subsets, were restimulated by IPCs, they proliferated and produced high amounts of IFN-gamma. These data indicate that IPCs can effectively stimulate preactivated or memory-type T cells and exert an immune-regulatory role. They also suggest that expansion of naive T cells and acquisition of effector function during antigen-specific T cell responses may involve different antigen-presenting cell (APC) types. Independent and coordinated control of T cell proliferation and differentiation would provide the immune system with greater flexibility in regulating immune responses.
Journal of Experimental Medicine 05/2003; 197(7):899-906. · 13.85 Impact Factor
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ABSTRACT: Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3epsilon Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3epsilon-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.
The Journal of Immunology 09/2002; 169(4):1837-43. · 5.79 Impact Factor
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ABSTRACT: Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.
The Journal of Immunology 07/2002; 168(12):6159-64. · 5.79 Impact Factor
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ABSTRACT: Immunization with superantigen in vivo induces transient activation of superantigen-specific T cells, followed by a superantigen-nonresponsive state. In this study, using a TCR alpha knock-in mouse in which the knock-in alpha-chain can be replaced with endogenous alpha-chain through secondary rearrangement, we show that immunization of superantigen changes the TCR alpha-chain expression on peripheral superantigen-specific T cells, induces expression of recombination-activating genes, and generates DNA double-strand breaks at the TCR alpha-chain locus. These results suggest that viral superantigens are capable of inducing peripheral TCR revision. Our findings thus provide a new perspective on pathogen-immune system interaction.
The Journal of Immunology 05/2002; 168(7):3259-65. · 5.79 Impact Factor
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ABSTRACT: We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.
The Journal of Immunology 03/2002; 168(3):1235-43. · 5.79 Impact Factor
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ABSTRACT: Using a TCRα chain knock-in mouse, we demonstrate that V-gene replacement can operate in the T cell receptor α locus. Functional TCRα chain transcripts generated by Vα-gene replacement at the site of the Vα-embedded heptamer were identified in splenic T cells. This finding shows that Vα-gene replacement can likely be used to shape the peripheral T cell repertoire.The conservation of the embedded heptamer in most Vα segments adds support to the notion that V-gene replacement is a mechanism maintained to diversify the immune system and that argues that itis common to B and T cells.
European Journal of Immunology 09/2001; 31(10):2919 - 2925. · 5.10 Impact Factor
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Science 08/1994; 265(5169):267. · 31.20 Impact Factor
