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Biomedical subjects

D H Raulet

Publications and source records attributed to D H Raulet.

At least 37 records · Page 2Linked to original sources
PubMed1999

Defective development of gamma/delta T cells in interleukin 7 receptor-deficient mice is due to impaired expression of T cell receptor gamma genes.

Mice lacking the interleukin 7 receptor (IL-7R) generate alpha/beta T cells at a detectable but greatly reduced rate, but gamma/delta T cells are completely absent. The special role of IL-7R signaling in gamma/delta T cell development has remained unclear. IL-7Ralpha(-/-) mice exhibit a paucity of gamma gene rearrangements. This striking observation can be explained by a defect in T cell receptor (TCR)-gamma gene rearrangement, a defect in TCR-gamma gene transcription leading to death of gamma/delta lineage cells, and/or a requirement for IL-7R in commitment of cells to the gamma/delta lineage. To determine the role of IL-7R signaling in gamma/delta T cell development, we examined transcription of a prerearranged TCR-gamma transgene in IL-7Ralpha(-/-) mice, as well as the effects of IL-7 on transcription of endogenous, rearranged TCR-gamma genes in alpha/beta lineage cells. The results demonstrate that IL-7R-mediated signals are necessary for the normal expression of rearranged TCR-gamma genes. Equally significant, the results show that the poor expression of TCR-gamma genes in IL-7Ralpha(-/-) mice is responsible for the selective deficit in gamma/delta cells in these mice, since a high copy TCR-gamma transgene exhibited sufficient residual expression in IL-7Ralpha(-/-) mice to drive gamma/delta cell development. The results indicate that the absence of gamma/delta T cells in IL-7Ralpha(-/-) mice is due to insufficient TCR-gamma gene expression.

Animals↗
PubMed1999

Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells.

The identity of cells that mediate positive selection of CD8(+) T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated beta(2)-microglobulin mutant mice or mice with mutations in both the K(b) and D(b) genes were repopulated with fetal liver cells from class I(+) TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8(+) T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8(+) T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8(+) T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)-TCR(+) CD8(+) T cells was observed in class I(+) into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.

Animals↗
PubMed1999

A novel element upstream of the Vgamma2 gene in the murine T cell receptor gamma locus cooperates with the 3' enhancer to act as a locus control region.

Transgenic expression constructs were employed to identify a cis-acting transcription element in the T cell receptor (TCR)-gamma locus, called HsA, between the Vgamma5 and Vgamma2 genes. In constructs lacking the previously defined enhancer (3'E(Cgamma1)), HsA supports transcription in mature but not immature T cells in a largely position-independent fashion. 3'E(Cgamma1), without HsA, supports transcription in immature and mature T cells but is subject to severe position effects. Together, the two elements support expression in immature and mature T cells in a copy number-dependent, position-independent fashion. Furthermore, HsA was necessary for consistent rearrangement of transgenic recombination substrates. These data suggest that HsA provides chromatin-opening activity and, together with 3'E(Cgamma1), constitutes a T cell-specific locus control region for the TCR-gamma locus.

Animals↗
PubMed1999

Development and tolerance of natural killer cells.

A natural killer (NK)/T-cell-restricted progenitor cell in the fetal blood, with a phenotype of NK1.1(+) CD117 (c-kit)+, can give rise to NK cells. IL-15 is probably required for NK cell differentiation and, in addition, stimulates expression of at least one of the MHC-specific inhibitory receptor families expressed by NK cells. Evidence supports the role of multiple mechanisms in rendering NK cells self tolerant - including selection for expression of self-specific inhibitory receptors, anergy and modulation of the cell surface levels of MHC-specific inhibitory receptors.

Animals↗
PubMed1999

Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors.

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

Alleles↗
PubMed1999

A new monoclonal antibody reactive with several Ly49 NK cell receptors mediates redirected lysis of target cells.

We produced a novel hamster monoclonal antibody (MAb), 14B11, that recognizes the majority of mouse natural-killer (NK) cells. Transfection studies demonstrated that 14B11 MAb binds a subset of Ly49 receptors, including three putative inhibitory receptors, Ly49F, I, and C. No binding to Ly49A, B, D, or G was detected. In addition, 14B11 was shown to bind the putative activating receptor Ly49H, which required co-transfection of the signaling molecule DAP12 for detectable cell surface expression. Thus, 14B11 is the first reported MAb to bind Ly49H and F. At the functional level, 14B11 MAb enhanced the lysis by IL-2 activated NK cells of an FcR+ target cell line (Daudi), but not an FcR- target cell (EL-4). Because F(ab')2 fragments of 14B11 failed to enhance lytic activity, the enhancement of lysis by intact antibody is apparently due to "redirected lysis," in which stimulatory receptors on the NK cell are bridged by antibody to Fc receptors on the target cell. Cell separation experiments demonstrated that the 14B11-dependent redirected lysis was markedly increased using NK cell populations that had been depleted of Ly49F,+ I,+ or C+ NK cells. Because such depletions are expected to enrich for Ly49H+ NK cells, these results suggest that the enhancement of lysis mediated by 14B11 MAb may be due to stimulation of the activating Ly49H receptor. In conjunction with other anti-Ly49 MAbs, the 14B11 MAb will be useful in further studies of Ly49 receptor function and specificity.

Animals↗
PubMed1998

Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b).

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.

Animals↗
PubMed1998

Acquisition of Ly49 receptor expression by developing natural killer cells.

The formation of the repertoire of mouse natural killer (NK) cell receptors for major histocompatibility complex (MHC) class I molecules was investigated by determining the developmental pattern of Ly49 receptor expression. During the first days after birth, few or no splenic NK cells express Ly49A, Ly49C, Ly49G2, or Ly49I receptors. The proportion of Ly49+ splenic NK cells gradually rises to adult levels during the first 6-8 wk of life. The appearance of appreciable numbers of splenic Ly49+ NK cells coincides with the appearance of NK activity at 3-4 wk. After in vivo transfer, NK cells not expressing specific Ly49 receptors can give rise to NK cells that do, and cells expressing one of these four Ly49 receptors can give rise to cells expressing others. Once initiated, expression of a Ly49 receptor is stable for at least 10 d after in vivo transfer. Hence, initiation of Ly49 receptor expression occurs successively. Interestingly, expression of one of the receptors tested, Ly49A, did not occur after in vivo transfer of Ly49A- cells. One possible explanation for these data is that the order of Ly49 receptor expression by NK cells is nonrandom. The results provide a framework for evaluating models of NK cell repertoire formation, and how the repertoire is molded by host class I MHC molecules.

Animals↗
PubMed1998

2F1 antigen, the mouse homolog of the rat "mast cell function-associated antigen", is a lectin-like type II transmembrane receptor expressed by natural killer cells.

Inhibitory lectin-like receptors expressed on the surface of hematopoietic cells are critically involved in regulation of their effector functions. Here we report that a novel mAb specific for mouse NK cells, 2F1, recognizes the mouse homolog of the mast cell function-associated antigen (MAFA), an inhibitory lectin-like transmembrane receptor expressed on rat mast cells. The 2F1 antigen (2F1-Ag) and rat MAFA are structurally highly conserved and contain a cytoplasmic motif similar to the immunoreceptor tyrosine-based inhibitory motif that is presumably utilized for inhibitory signaling. We also identified a human homolog that is closely related to the rodent MAFA/2F1-Ag proteins. Like rat MAFA, 2F1-Ag is probably encoded by a single gene, which exhibits relatively little polymorphism. Strikingly, while rat MAFA is considered a mast cell antigen, we have been unable to detect cell surface expression of 2F1-Ag by mouse mast cell lines, bone marrow-derived mast cells, or peritoneal mast cells. Furthermore, mouse bone marrow-derived mast cells were devoid of 2F1-Ag mRNA. Instead, we find that approximately 40% of mouse NK cells express 2F1-Ag. Thus, MAFA/2F1-Ag may modulate immunological responses on at least two different cell types bridging the specific and innate immune system.

Amino Acid Sequence↗
PubMed1998

The developmental fate of T cells is critically influenced by TCRgammadelta expression.

Differentiation of gammadelta and alphabeta T cells from a common precursor cell depends on productive rearrangement and expression of TCRgammadelta or TCRbeta genes, but whether it is an instructive or a stochastic mechanism that is responsible for this process is unclear. We report that expression of the productively rearranged TCRgamma transgene competitively inhibits alphabeta thymocyte development under conditions where TCRbeta gene rearrangement is limiting. The status of TCRdelta gene rearrangements in the remaining alphabeta-lineage cells indicates that the effect is mediated by the intact gammadelta receptor. Paradoxically, in TCRbeta-/- mice, gammadelta receptor expression can also drive differentiation of some alphabeta-lineage cells. To resolve this paradox, we provide evidence for a minor population of gammadelta-dependent alphabeta-lineage cells in normal mice. The results indicate that the T cell lineage commitment process is either error-prone or stochastic.

Animals↗
PubMed1998

T cell receptor gamma gene regulatory sequences prevent the function of a novel TCRgamma/pTalpha pre-T cell receptor.

Expression of a TCRgamma transgene in RAG-1-/- mice resulted in the development of a limited number of CD4+CD8+ (DP) thymocytes. In vivo treatments with anti-TCRgamma antibody enhanced the number of DP thymocytes, demonstrating that TCRgamma chains were expressed on the cell surface in the absence of delta, alpha, or beta chains. Mutations in pTalpha or CD3epsilon genes abolished transgene-induced DP cell development, indicating that TCRgamma can associate with pTalpha and CD3 to form a novel pre-TCR. With a transgene containing additional regulatory sequences, TCRgamma expression was down-regulated in DP cells, and little DP cell development occurred. Thus, the function of the endogenous TCRgamma/pTalpha is limited by the transcriptional down-regulation of TCRgamma genes that normally accompanies DP cell development.

Animals↗
PubMed1998

Developmentally programmed rearrangement of T cell receptor Vgamma genes is controlled by sequences immediately upstream of the Vgamma genes.

Distinct subsets of gammadelta T cells expressing different Vgamma and Vdelta chains arise in ordered waves during thymic development. In the murine Jgamma1-Cgamma1 cluster, the Vgamma3 gene segment is utilized earliest in fetal thymic development, in progenitors of dendritic epidermal T cells (DECs). The Vgamma2 gene segment predominates in the late fetal stages and beyond, in cells destined for the secondary lymphoid organs. Using transgenic TCRgamma recombination substrates, we demonstrate that this restricted Vgamma gene usage is determined by developmentally targeted gene rearrangement. We show that sequences immediately upstream of the Vgamma2 and Vgamma3 genes direct the rearrangement pattern in adult thymocytes. Thus, the choice of Vgamma gene for recombination is coordinated with distinct differentiation programs in gammadelta subsets.

Animals↗
PubMed1997

The basis for self-tolerance of natural killer cells in beta2-microglobulin- and TAP-1- mice.

Cells from mice with mutations in the genes for beta2-microglobulin (beta2m) or for TAP-1 express only low levels of MHC class I proteins on their surfaces, and are thus sensitive to attack by normal NK cells. Although NK cells are present in beta2m- mice and TAP-1(-) mice, they are completely self-tolerant. The underlying mechanism for this tolerance is unknown. It has been proposed that education processes render NK cells from these mice hypersensitive to class I-mediated inhibition, so that they can be inhibited even by the low levels of class I expressed on autologous cells. In this study, we present evidence against this hypothesis, by demonstrating that NK cells from beta2m- mice and TAP-1(-) mice fail to attack beta2m(-)TAP-1(-) double-mutant cells in both in vitro and in vivo assays. The latter cells express substantially lower levels of class I than single-mutant cells, based on serologic tests, as well as a significantly diminished sensitivity to attack by class I-specific CTL. Furthermore, the Ly-49 repertoire on NK cells derived from beta2m(-)TAP-1(-) mice is highly similar to that of either single mutant, indicating that the developmental processes that shape the Ly-49 repertoire cannot respond to the differences in class I levels among these mice. We propose that self-tolerance of NK cells in beta2m- mice and TAP-1(-) mice is likely to result from hyporesponsiveness of the cells to activating signals, or alternatively, to induction of inhibitory signaling through receptors specific for non-class I MHC ligands.

Animals↗
PubMed1997

Ly49A transgenic mice provide evidence for a major histocompatibility complex-dependent education process in natural killer cell development.

The Ly49 natural killer (NK) cell receptors are class I MHC-specific inhibitory receptors that are distributed to overlapping NK cell subsets. The formation of the Ly49 receptor repertoire was examined with transgenic mice that express Ly49A in all NK cells. In MHC class I-deficient mice, the Ly49A transgene did not prevent expression of endogenous Ly49 genes. However, in H-2(d) mice that express a Ly49A ligand, the transgene caused clear alterations in the endogenous Ly49 repertoire. The frequency of NK cells expressing another H-2(d)-specific receptor, Ly49G2(+), was substantially reduced. Reduced numbers of cells expressing endogenous Ly49A was suggested by reduced endogenous Ly49A mRNA levels. These results support the existence of an MHC-dependent education process that limits the number of NK cells that coexpress multiple self-specific Ly49 receptors. Ligand-dependent downregulation of Ly49 cell surface levels was also examined. Cell-surface downregulation occurred even when the transgene was expressed at low levels. The results demonstrate that downregulation of Ly49A cell surface levels is a posttranscriptional event, and argue against a model in which Ly49 receptors are calibrated to specific cell surface levels depending on the available class I ligands.

Animals↗
PubMed1997

The MHC reactivity of the T cell repertoire prior to positive and negative selection.

T cell antigen receptors (TCRs) on mature T cells react with peptide antigens presented by self-MHC proteins and also frequently cross-react with foreign MHC proteins. The fundamental question whether MHC reactivity is inherent in the germline TCR sequences or is imposed by thymic selection was addressed here by inducing nonselective maturation of immature thymocytes in the absence of MHC molecules. MHC reactivity in the preselection repertoire is very high, but no higher than in the normal repertoire. Cross-reactivity of clones with multiple MHC molecules occurred to a similar extent in the preselection and MHC-selected repertoires. The results establish the MHC reactivity of the germline TCR repertoire, indicate the minimum fraction of immature thymocytes that must undergo negative selection, and suggest that some TCR-MHC contacts may be conserved.

Animals↗
PubMed1997

Class I-deficient hemopoietic cells and nonhemopoietic cells dominantly induce unresponsiveness of natural killer cells to class I-deficient bone marrow cell grafts.

NK cells in normal mice reject bone marrow transplants from class I-deficient mice. In contrast, class I-deficient mice do not reject autologous cells, suggesting that NK cell tolerance is acquired. We employed fetal liver irradiation chimeras to assess two potential mechanisms that could account for the tolerance of NK cells in class I-deficient mice to class I-deficient cells: 1) a positive model, in which recognition of class I+ cells molecules by NK cells is necessary to induce functional NK cell maturation; and 2) a negative model, in which interactions of NK cells with class I-deficient cells induce tolerance. In class I+ chimeras reconstituted with mixtures of class I+ and class I-deficient fetal liver cells, the rejection of class I-deficient bone marrow cell grafts was significantly impaired, supporting the negative model. We further addressed whether nonhemopoietic cells are also able to induce NK cell tolerance. Class I- mice reconstituted with class I+ fetal liver cells were tolerant of class I-deficient cells, favoring this idea. Furthermore, class I-deficient mice reconstituted with a mixture of class I-deficient and class I+ fetal liver cells were more tolerant to class I-deficient cells than were class I+ mice reconstituted with the same fetal liver cell mixture. These results suggest that maximal tolerance induction requires the presence of class I-deficient nonhemopoietic cells.

Animals↗