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D H Raulet

Publications and source records attributed to D H Raulet.

At least 91 records · Page 5Linked to original sources
PubMed1989

Positive selection determines T cell receptor V beta 14 gene usage by CD8+ T cells.

We report here a mAb, 14-2, reactive with TCRs that include V beta 14. The frequency of V beta 14+ T cells varies with CD4 and CD8 subset and is controlled by the H-2 genes. Thus CD8+ T cells from H-2b mice include approximately 2.3% V beta 14+ T cells while CD8+ T cells from mice expressing K kappa include greater than 8% V beta 14+ T cells. In all strains examined, 7-8% of CD4+ T cells express V beta 14. The frequent usage of V beta 14 in CD8+ T cells of K kappa-expressing mice is a result of preferential positive selection of V beta 14+ CD8+ T cells as demonstrated by analysis of radiation chimeras. These studies demonstrate that H-2-dependent positive selection occurs in unmanipulated mice. Furthermore, the results imply that positive selection, and possibly H-2 restriction, can be strongly influenced by a V beta domain, with some independence from the beta-junctional sequence and alpha chain.

Animals↗
PubMed1989

A novel gamma delta T cell receptor for antigen adds limited diversity to the gamma delta repertoire in adult thymus.

The surface expression of CD3-associated TCR chains on hybridoma cell lines derived from adult gamma delta thymocytes was analyzed. These cell lines were unusual, in that a) they expressed a surface heterodimer consisting of a 40- and a 42-kDa chain, i.e., comprised of chains different from any previously reported gamma delta-TCR all of which express C gamma 1- or C gamma 2-encoded gamma-chains; b) their CD3-associated TCR could not be categorized as alpha beta-TCR dimers, despite the similarities in m.w. of the TCR chains, because full size 1.3-kb beta-chain mRNA capable of encoding a functional beta-chain could not be detected in these cells; c) neither of the receptor chains could be precipitated with anti-C gamma 1C gamma 2-peptide antisera. Biochemical analysis demonstrated that the 42-kDa delta-chain is a novel chain, which differs from any reported delta-chains in size, charge and number of glycosylation sites. Collectively, the data on analysis of the 40-kDa chain strongly suggest that it represents a gamma-chain encoded for by the C gamma 4 locus, protein products of which have not yet been reported in the thymus. This gamma-chain was also unique, in that its isoelectric point was much lower than that of other gamma-chains. The gamma- and delta-chains on these C gamma 4-expressing hybridomas were indistinguishable from one another in size and charge (as determined by nonequivalent pH gradient electrophoresis/SDS-PAGE analysis and analysis after endoglycosidase treatment). Because the cell lines were randomly chosen from large panels of hybridomas, these results may well imply strikingly nonrandom pairing of thymocyte-derived C gamma 4 chains and the delta-chains reported here. Thus, only limited additional gamma delta repertoire diversity may be generated by availability of this gamma delta-TCR in the thymus.

Aging↗
PubMed1989

Rearrangement by inversion of a T-cell receptor delta variable region gene located 3' of the delta constant region gene.

We have located a T-cell receptor variable (V) delta gene segment immediately 3' of the delta constant (C) region gene and 5' to the known joining (J) alpha gene segments. This V delta gene is in the opposite transcriptional polarity to C delta and has rearranged to C delta by inversion in a gamma/delta-expressing hybridoma, DN7.3. This V delta gene is commonly rearranged in adult but not fetal gamma/delta-expressing thymocytes and has not been observed among alpha gene rearrangements reported to date. The reciprocal joining sequence isolated from this cell line contains N region nucleotides between the recombination signal sequences, in contrast to previously analyzed reciprocal joints. The results are discussed in the context of models accounting for ordered V gene usage during lymphocyte development.

Amino Acid Sequence↗
PubMed1988

Predominant variable region gene usage by gamma/delta T cell receptor-bearing cells in the adult thymus.

Previous studies have indicated that the diversity of gamma genes expressed by gamma/delta-bearing murine T cells is limited, but comparable information concerning the expressed diversity of delta genes is lacking. In this study, we have investigated the rearrangement and expression of delta and gamma genes in T cell hybridomas that express gamma/delta T cell receptors. Three productive delta chain cDNA clones were isolated (delta 7.3, delta 7.1, and delta 2.3) that encode new variable region sequences. Two of the delta cDNAs differ significantly from those observed in the V alpha repertoire. In addition, one cDNA expressed a new J delta region (J delta 2), which was localized between J delta 1 and C delta genes. Using these and other delta gene probes and gamma gene probes, we found that five independent hybridomas expressed four different V delta s and three different V gamma s. However, analysis of an enriched population of gamma/delta-expressing cells from the adult thymus suggests that only a few V delta genes and one V gamma gene are used by the majority of the cells. These results suggest that important components of receptor chain that contribute to specificity (i.e., the germline V gene sequences) are relatively nondiverse in the thymic gamma/delta population.

Amino Acid Sequence↗
PubMed1987

Characterization of a novel murine T cell-activating factor.

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.

Animals↗
PubMed1987

B-cell-stimulatory factor 2 (beta 2 interferon) functions as a second signal for interleukin 2 production by mature murine T cells.

Purified peripheral murine T cells, in the presence of concanavalin A, can be activated to produce interleukin 2 (IL-2) through stimulation either with a previously described murine lymphokine designated T cell-activating factor (TAF) or with a cloned human lymphokine that has been called beta 2 interferon, B-cell-stimulatory factor 2, hybridoma growth factor, inducible 26-kDa protein, or hematopoietic colony-stimulating factor 309 by different investigators. We and others propose the designation interleukin 6 (IL-6) for the latter molecule. Our experiments demonstrate that either murine TAF or human IL-6 can restore the ability of purified T cells to proliferate in response to Con A or antibodies against the T-cell antigen receptor. Most if not all of the proliferation can be blocked by antibodies against the alpha chain of the IL-2 receptor. Furthermore, highly purified CD8- T cells can be activated by IL-6 in the presence of Con A to secrete IL-2. We propose that IL-6 and murine TAF are important "second signals" in primary antigen-receptor-dependent T-cell activation. Whether or not murine TAF is a homologue of human IL-6 remains to be determined.

Animals↗
PubMed1986

Developmental potential and dynamic behavior of hematopoietic stem cells.

We have used retrovirus-mediated gene transfer to mark hematopoietic stem cells in vitro and have tracked the fate of these cells after their transplantation into lethally irradiated recipients. Several classes of stem cells are demonstrated, including cells whose progeny completely repopulate all hematopoietic lineages as well as cells that contribute predominantly to certain lineages or to specific anatomical locations. In a majority of recipients, we find that few (1 or 2) stem-cell clones account for the majority of the mature hematopoietic cells. These results coupled with retransplantation studies suggest an in vivo mechanism for the temporal control of stem-cell use. Further studies based on periodic sampling of primary recipients suggest that normal hematopoiesis results from the sequential activation of different stem-cell clones rather than from an averaged contribution of the entire stem-cell pool.

Animals↗
PubMed1986

Diversity, rearrangement, and expression of murine T cell gamma genes.

Although the T cell gamma genes are similar in many respects to T cell receptor alpha and beta genes, earlier studies suggested that only a single gamma variable (V gamma) gene is expressed in mature T cells. We report the isolation and characterization of three new rearranged V gamma genes from murine fetal thymocytes. Although each of the new V gamma gene rearrangements is present in fetal thymocytes, two of them are undetectable in mature T cells. The levels of mRNA corresponding to each type of V gamma gene rearrangement in mature T cells are dramatically diminished compared with those in fetal thymocytes, although the abundance of two of the rearranged genes is increased in mature T cells. Our results demonstrate that there is significant expressed variability of gamma genes in immature T cells. Furthermore, the dynamics of gamma gene rearrangement and expression support the idea that gamma genes function in immature T cells.

Animals↗
PubMed1986

T-cell receptor variable region gene usage in T-cell populations.

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.

Animals↗
PubMed1986

A phosphorylated, disulfide-linked membrane protein in murine cytotoxic T lymphocytes.

The previously determined sequence of the murine T-cell gamma gene and its transcription in cloned T lymphocytes suggests that the polypeptide encoded by this gene is generally present in cytotoxic T cells as a 33-kDa monomer in a disulfide-bonded dimer. The gamma chain is also expected to be phosphorylated because a sequence in its cytoplasmic domain is homologous to an active site for serine phosphorylation in the regulatory subunit of cAMP-dependent protein kinase. We describe here a cytotoxic-T-cell-associated phosphorylated protein, many of whose properties suggest that it may be the product of the T-cell gamma gene. Its phosphorylation is greatly enhanced by interleukin 2 stimulation.

Amino Acids↗
PubMed1982

Helper T cells for cytotoxic T lymphocytes need not be I region restricted.

We investigated the antigenic requirements for restimulation of H-2- restricted cytolytic T lymphocytes (CTL) in vitro to determine whether H-2 I region-restricted helper T cells are required in these responses. In one set of experiments, we studied the in vitro response of (responder x nonresponder)F(1) female T cells to the male antigen H-Y. We chose to examine this response because it has been suggested that the defect in nonresponder strains is a failure of helper T cells to recognize H-Y in association with nonresponder I region determinants. However, we find that nonresponder male stimulator cells are as effective as F(1) male stimulator cells at inducing H-Y-specific CTL responses. This finding calls into question reports that secondary CTL responses to H-Y are dependent upon the activation of H-Y- specific helper T cells restricted to responder type I region determinants. In a second set of experiments, we examined the requirements for restimulation of H-2-restricted T cells specific for minor-histocompatibility antigens from long-term mixed lymphocyte cultures. These cultures were established by repeatedly restimulating cultures of specific T cells with H- 2-matched stimulator cells expressing foreign minor histocompatibility antigens. We found that H-2D-restricted T ceils, including CTL, could be restimulated with cells that were matched with the responding cells at only the D region genes. This response did not appear to result from positive allogeneic effects or from antigen processing and "representation" by responder type APC that might contaminate the cultures. Thus, we find no evidence for a requirement for I region-restricted helper T cells in these CTL responses. However, helper T cells are required because we find that CTL lines derived by limit-dilution cloning from these long-term MLC are absolutely dependent upon exogenous helper factors for growth. The most simple interpretation of these results is that the helper cells are restricted to H-2 antigens other than I region antigens or to antigens that code outside of the H-2 complex. Finally, we show that factor-dependent CTL lines must recognize their specific antigen to proliferate, even in the presence of exogenous factors. The requirement of activated CTL for antigen to proliferate provides an explanation for how specific CTL can be selectively enriched in MLC by specific antigen stimulation. Furthermore, it is at variance with reports that memory CTL or activated CTL require only interleukin 2 for restimulation.

Animals↗